U.S. patent application number 14/894043 was filed with the patent office on 2016-04-28 for self-assembled micro-and nanostructures.
This patent application is currently assigned to RAMOT AT TEL-AVIV UNIVERSITY LTD.. The applicant listed for this patent is NORTHWESTERN UNIVERSITY, RAMOT AT TEL-AVIV UNIVERSITY LTD.. Invention is credited to Lihi ADLER-ABRAMOVICH, Galit FICHMAN, Ehud GAZIT, Phillip B. MESSERSMITH.
Application Number | 20160115196 14/894043 |
Document ID | / |
Family ID | 51134165 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160115196 |
Kind Code |
A1 |
FICHMAN; Galit ; et
al. |
April 28, 2016 |
SELF-ASSEMBLED MICRO-AND NANOSTRUCTURES
Abstract
The present invention discloses self-assembled bioadhesive
anti-microbial, anti-fouling and/or anti-oxidant micro- and
nano-structures comprising a plurality of amino acids or peptides,
wherein each amino acid is an aromatic amino acid comprising a
catecholic moiety, and/or each peptide comprises at least one
aromatic amino acid comprising a catecholic moiety. Further
disclosed are methods and kits for preparing these micro- and
nano-structures. Further disclosed are uses of these micro- and
nano-structures in pharmaceutical, cosmetic and medical devices
applications.
Inventors: |
FICHMAN; Galit; (Yavne,
IL) ; ADLER-ABRAMOVICH; Lihi; (Herzeliya, IL)
; GAZIT; Ehud; (Ramat Hasharon, IL) ; MESSERSMITH;
Phillip B.; (Clarendon Hills, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NORTHWESTERN UNIVERSITY
RAMOT AT TEL-AVIV UNIVERSITY LTD. |
Evanston
Tel Aviv |
IL |
US
IL |
|
|
Assignee: |
RAMOT AT TEL-AVIV UNIVERSITY
LTD.
Tel Aviv
IL
|
Family ID: |
51134165 |
Appl. No.: |
14/894043 |
Filed: |
May 28, 2014 |
PCT Filed: |
May 28, 2014 |
PCT NO: |
PCT/IL2014/050479 |
371 Date: |
November 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61827785 |
May 28, 2013 |
|
|
|
Current U.S.
Class: |
514/2.4 ;
428/34.1; 428/402; 514/21.8; 514/21.9; 514/21.91; 530/330; 530/331;
562/448 |
Current CPC
Class: |
A61K 38/00 20130101;
A01N 37/46 20130101; C07K 17/04 20130101; C07K 5/0812 20130101;
C07K 5/06078 20130101; C07K 7/06 20130101 |
International
Class: |
C07K 5/065 20060101
C07K005/065; C07K 7/06 20060101 C07K007/06; A01N 37/46 20060101
A01N037/46; C07K 5/087 20060101 C07K005/087 |
Claims
1-41. (canceled)
42. A self-assembled micro- or nano-structure comprising (i) a
plurality of aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA) and a DOPA-derivative; or (ii)
a plurality of peptides, each peptide comprising between 2 and 9
amino acids, at least one of which is an aromatic amino acid
selected from 3,4-dihydroxyphenyl-L-alanine (DOPA) and a
DOPA-derivative; or (iii) a combination of said amino acids and
peptides; wherein said micro- or nano-structure has at least one
property selected from bioadhesive, anti-oxidant, anti-fouling,
anti-bacterial and any combination thereof.
43. The micro- or nano-structure of claim 42, which is selected
from the group consisting of a fibrillar micro- or nano-structure,
a tubular micro- or nano-structure, a spherical micro- or
nano-structure and a ribbon-like micro- or nano-structure.
44. The micro- or nano-structure of claim 43, which is at least
about 1 nm in diameter, and which does not exceed about 500 nm in
diameter.
45. The micro- or nano-structure of claim 42, wherein each peptide
in said plurality of peptides comprises between 2 and 7 amino
acids.
46. The micro- or nano-structure of claim 42, comprising a
combination of said amino acids and said peptides.
47. The micro- or nano-structure of claim 42, wherein each peptide
in said plurality of peptides comprises a plurality of aromatic
amino acids selected from 3,4-dihydroxyphenyl-L-alanine (DOPA), a
DOPA-derivative and a combination thereof.
48. The micro- or nano-structure of claim 42, wherein at least one
peptide in said plurality of peptides is a
3,4-dihydroxyphenyl-L-alanine-(3,4-dihydroxyphenyl-L-alanine)
(DOPA-DOPA) homodipeptide.
49. The micro- or nano-structure of claim 42, wherein at least one
peptide in said plurality of peptides incorporates at least one
3,4-dihydroxyphenyl-L-alanine-(3,4-dihydroxyphenyl-L-alanine)
(DOPA-DOPA) homodipeptide in the peptide backbone.
50. The micro- or nano-structure of claim 48, wherein said
DOPA-DOPA homopeptide further comprises at least one end-capping
modified moiety at the C- or N-terminus.
51. The micro- or nano-structure of claim 42, wherein at least one
amino acid or peptide in said plurality of amino acids or peptides
further comprises at least one amino acid capable of enhancing
cohesion, enhancing adhesion of said peptide to a surface, or a
combination thereof.
52. The micro- or nano-structure of claim 51, wherein said amino
acid is charged at neutral pH, wherein said amino acid comprises a
positively charged side chain capable of ionically interacting with
negatively charged surface, or a negatively charged side chain
capable of ionically interacting with positively charged
surface.
53. The micro- or nano-structure of claim 52, wherein said amino
acid is selected from the group consisting of lysine, ornithine,
arginine, aspartic acid, glutamic acid, and histidine.
54. The micro- or nano-structure of claim 42, wherein at least one
amino acid or peptide in said plurality of amino acids or peptides
comprises at least one end-capping modified moiety at the C- or
N-terminus.
55. The micro- or nano-structure of claim 54, wherein said end
capping moiety is selected from the group consisting of an aromatic
end capping moiety and a non-aromatic end-capping moiety.
56. The micro- or nano-structure of claim 55, wherein said aromatic
end capping moiety is selected from the group consisting of
9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz),
naphthalene (Nap) derivatives, phenothiazine (PTZ), azobenzene
(Azo), pyrene (Pyr), and cinnamoyl.
57. The micro- or nano-structure of claim 55, wherein said
non-aromatic end capping moiety is selected from the group
consisting of acetyl and tert-butoxycarbonyl (Boc).
58. The micro- or nano-structure of claim 54, wherein said
end-capping moiety comprises a labeling moiety.
59. The micro- or nano-structure of claim 42, wherein at least one
of the plurality of amino acids or peptides is selected from the
group consisting of Fmoc-DOPA, DOPA-DOPA, DOPA-Phe-Phe,
Fmoc-DOPA-DOPA, Fmoc-DOPA-DOPA-Lys, Fmoc-Phe-Phe-DOPA-DOPA-Lys,
Lys-Leu-Val-DOPA-DOPA-Ala-Glu, Asp-DOPA-Asn-Lys-DOPA and
derivatives of any of the foregoing comprising an end capping
moiety, preferably an Fmoc moiety.
60. The micro- or nano-structure of claim 42, which comprises: (i)
a plurality of aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA), a DOPA-derivative and
combinations thereof; or a plurality of peptides comprising at
least one aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA), a DOPA-derivative and
combinations thereof; (ii) at least one amino acid which is charged
at neutral pH; and (iii) optionally, at least one additional amino
acid selected from the group consisting of naturally occurring
amino acids, synthetic amino acids and combinations thereof,
wherein said micro- or nano-structure is bio-adhesive.
61. The micro- or nano-structure of claim 42, which is provided in
the form of a hydrogel.
62. The micro- or nano-structure of claim 42, which is co-assembled
with one or more additional self-assembled peptides, polypeptides,
polysaccharides, polymers, or a combination thereof.
63. The micro- or nano-structure of claim 62, wherein the
additional self-assembled peptide is selected from Phe-Phe and Fmoc
Phe-Phe, wherein Phe is phenylalanine.
64. A method of generating the self-assembled micro- or
nano-structure of claim 1, the method comprising incubating a
plurality of amino acids or peptides under conditions which favor
formation of said micro- and nano-structure.
65. The micro- or nano-structure of claim 42, for use in
preparation of pharmaceutical composition, cosmetic composition, a
medical device or a medical device coating.
66. The micro- or nano-structure of claim 65, for use in
preparation of biological glue.
67. The micro- or nano-structure of claim 65, for use as an
anti-oxidant, a radical trapper, a metal chelator, or an oxidizable
reducing agent.
68. A method of combating bacteria, comprising the step of
contacting the bacteria with the micro- or nano-structure of claim
42.
69. A method of disinfecting a surface, comprising the step of
contacting said surface with the micro- or nano-structure of claim
42.
70. A pharmaceutical composition, cosmetic composition, a medical
device or a medical device coating, comprising the self-assembled
micro- or nano-structure of claim 42, and a pharmaceutically or
cosmetically acceptable carrier.
71. A kit for forming the self-assembled micro- or nano-structure
of claim 42, the kit comprising (i) a plurality of aromatic amino
acids selected from 3,4-dihydroxyphenyl-L-alanine (DOPA) and a
DOPA-derivative; or a plurality of peptides, each peptide
comprising between 2 and 9 amino acids, at least one of which is an
aromatic amino acid selected from 3,4-dihydroxyphenyl-L-alanine
(DOPA) and a DOPA-derivative; or a combination of said amino acids
and peptides and (ii) an aqueous solution, each being individually
packaged within the kit, wherein said plurality of amino acids or
peptides and said solution are selected such that upon contacting
said plurality of peptides and said solution, said micro- or
nano-structure is formed.
72. A composition comprising the micro- or nano-structure of claim
42, and an agent selected from the group consisting of a
therapeutically active agent, a diagnostic agent, a biological
substance and a labeling moiety; wherein the micro- or
nano-structure optionally encapsulates said agent, or is attached
to said agent.
73. The composition according to claim 72, wherein the agent is
selected from the group consisting of drugs, cells, proteins,
enzymes, hormones, growth factors, nucleic acids, organisms such as
bacteria, fluorescence compounds or moieties, phosphorescence
compounds or moieties, and radioactive compounds or moieties.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to self-assembled bioadhesive,
anti-microbial, anti-fouling and/or anti-oxidant micro- and
nano-structures comprising a plurality of amino acids or peptides,
the micro- or nano-structures comprising at least one aromatic
amino acid comprising a catecholic moiety. The present invention
further relates to methods of preparing the self-assembled micro-
and nano-structures and to their use in a variety of biomedical and
industrial applications, for example in pharmaceutical and cosmetic
compositions and in medical devices.
BACKGROUND OF THE INVENTION
[0002] Bioadhesives are natural polymeric materials with adhesive
properties. Bioadhesives may be comprised of a variety of
substances, but proteins and carbohydrates feature prominently.
Several types of bioadhesives offer adhesion in wet environments
and under water, while others can stick to low surface
energy--non-polar surfaces, such as plastic. However, the most
prominent use of bioadhesives due to the biocompatibility thereof
is in biomedical applications, for example in the preparation of
biological glues. Current biological glues (fibrin, albumin,
gelatin-resorcinol-formaldehyde, etc.) suffer from low bond
strength and are in some cases derived from blood products, with
associated risk of viral or prion contamination. On the other hand,
synthetic glues (e.g., cyanoacrylate adhesives) are very strong but
they are also toxic to living tissues and form rigid, nonporous
films that can hinder wound healing [1].
[0003] Natural adhesion of mussels, other bivalves and algae to
rocks and other substrates has been described, for example, in U.S.
Pat. No. 5,015,677, U.S. Pat. No. 5,520,727 and U.S. Pat. No.
5,574,134. The adhesion of marine mussels has drawn a particular
interest due to their remarkable ability to bind strongly to
virtually all inorganic and organic surfaces in wet environment
under the conditions in which most adhesives function poorly [2].
In recent studies it was found that every type of mussel adhesive
protein (MAP) contains 5-30% of the non-coded amino acid
3,4-dihydroxyphenyl-L-alanine (DOPA), which is obtained by
hydroxylation of tyrosine residues. Moreover, the DOPA content in
the protein was found to correlate with the protein adhesive
strength, such that MAPs, which exhibit strong adhesion and are
typically found close to the adhesion interface, comprise a higher
proportion of DOPA residues [3]. The exact role of DOPA in MAPs is
not fully understood. Nevertheless, the catechol functionality of
DOPA residues is thought to be responsible for both cross-linking
and adhesion of the MAPs, as it can form several chemical
interactions including hydrogen bonding, metal-ligand complexation,
Michael-type addition, .pi.-.pi. interactions and quinhydrone
charge-transfer complexation [4].
##STR00001##
3,4-dihydroxyphenyl-L-alanine (DOPA)
[0004] EP Patent No. 1589088 is directed to biodegradable
compositions comprising adhesive, biocompatible polymers and
methods used to cover surfaces and to attach structures to eye
tissues, such as the cornea. Polyphenolic proteins isolated from
mussels (MAPs) are used in conjunction with polysaccharides and
pharmaceutically acceptable fine filaments, to achieve strong
adhesive bonding.
[0005] PCT Patent Application No. WO 2006/038866 discloses an
improved coating for biomedical surfaces including a bioadhesive
polyphenolic protein derived from a byssus-forming mussel, e.g.
Mefp-1 (Mytilus edulis foot protein-1).
[0006] Extraction of MAPs from mussels is, however, not practical
for commercial scale production. Attempts to mimic mussels'
adhesion properties were made, typically using synthetic or
genetically engineered polypeptides containing amino acid motifs
derived from mussel adhesives, or by incorporating DOPA into
synthetic polymers.
[0007] U.S. Pat. No. 4,908,404 discloses a water soluble cationic
peptide-containing graft copolymer exhibiting a number average
molecular weight of from about 30,000 to about 500,000 comprising:
(a) a polymeric backbone containing or capable of modification to
include free primary or secondary amine functional groups for
reaction with an amino acid or peptide graft and exhibiting a
number average molecular weight from about 10,000 to about 250,000;
and (b) an amino acid or peptide graft reacted with from at least
about 5% to about 100% of the primary or secondary amine functional
groups of the polymeric backbone, wherein said amino acid or
peptide graft comprises at least one 3,4-dihydroxyphenylalaine
(DOPA) amino acid or a precursor thereof capable of hydroxylation
to the DOPA form.
[0008] U.S. Patent Application No. 2005/0201974 is directed to
polymers with improved bioadhesive properties and to methods for
improving bioadhesion of polymers, wherein a compound containing an
aromatic group which contains one or more hydroxyl groups is
grafted onto a polymer or coupled to individual monomers, and
wherein, in a preferred embodiment, the polymer is a polyanhydride
and the aromatic compound is the catechol derivative, DOPA.
[0009] EP Patent Application No. 0242656 discloses methods for
forming bioadhesive polyphenolic proteins containing
3,4-dihydroxyphenylalanine residues from protein precursors
containing tyrosine residues.
[0010] U.S. Patent Application No. 2003/0087338 to one of the
inventors of the present invention is directed to a route for the
conjugation of DOPA moieties to various polymeric systems,
including poly(ethylene glycol) or poly(alkylene oxide) systems
such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene
oxide) (PEO-PPO-PEO) block copolymers.
[0011] Additional polymers that are end functionalized with DOPA
groups are described in scientific literature [5-8]. Furthermore,
Statz et. al. describe DOPA-modified oligomeric peptides that were
developed to generate stable antifouling surface coatings [9].
[0012] A key element in future nanotechnology is the use of
nanostructures fabricated through molecular self-assembly [10]. In
a bottom-up process, simple building blocks self-assemble to form
large and more complex supramolecular assemblies. In the molecular
self-assembly process, molecules spontaneously interact with each
other through noncovalent bonds, such as Van der Waals
interactions, hydrogen bonds, aromatic interactions and
electrostatic interactions, to form well-ordered ultrastructures.
In recent years there is a great interest in the fabrication of new
materials using natural building blocks and combining them in
artificial systems, when proteins and peptides are of special
interest due to their biological and chemical diversity as well as
structural simplicity.
[0013] As has been previously shown by some of the inventors of the
present invention, interactions between aromatic units play a
central role in molecular self-assembly of amino acids [11-14].
These are attractive non-covalent interactions between planar
aromatic rings which are referred as .pi.-stacking. Aromatic
interactions are made up of a combination of forces including
electrostatic, hydrophobic and Van der Waals interactions. A major
characteristic of aromatic interactions is their specific
geometries, which endow them with specific properties, enabling
recognition and selectivity. Reductionist approaches have shown
that aromatic tetrapeptide fragments self-assemble to form
amyloid-like structures [15]. In addition, the core recognition
motif of the .beta.-amyloid polypeptide, which plays a key role in
Alzheimer disease, the diphenylalanine, has been shown to form
ordered tubular and spherical structures [14]. Later studies
reveled that other aromatic homodipeptides could form various
structures at the nano-scale, including nanotubes, nanospheres,
fibrillar assemblies, nano-plates and hydrogels.
[0014] U.S. Pat. No. 7,786,086 to some of the inventors of the
present invention discloses a nanostructure composed of a plurality
of peptides, each peptide containing at least one aromatic amino
acid, whereby one or more of these peptides is end-capping modified
and wherein the nanostructure can take a tubular, fibrillar, planar
or spherical shape, and can encapsulate, entrap or be coated by
other materials.
[0015] U.S. Patent Application No. 2009/0175785 to some of the
inventors of the present invention is directed to novel
peptide-based hydrogels, composed of short aromatic peptides (e.g.,
homodipeptides of aromatic amino acid residues).
[0016] EP Patent No. 1575867 to some of the inventors of the
present invention discloses a tubular or spherical nanostructure
composed of a plurality of peptides, wherein each of the plurality
of peptides includes no more than 4 amino acids and whereas at
least one of the 4 amino acids is an aromatic amino acid.
[0017] The major disadvantage of presently known bioadhesive
materials, mimicking mussel adhesive properties, is the
uncontrollable presentation of the functional adhesive sites
relatively to the surface. In order to exploit the bioadhesive
properties of nonoxidized DOPA-functionalized materials, it is
important to have control over the presentation of the functional
adhesive sites relative to the surface. Currently known
DOPA-functional synthetic polymers expose the adhesive groups
randomly, which reduces the adhesive properties of
DOPA-functionalized materials.
[0018] The modification of polymers with catechol groups draws
significant attention beyond adhesion due to the abilities of these
groups to act as antioxidant agents, radical trappers, metal
chelators, oxidizable reducing agents, etc. [25, 26]. Moreover,
catechol redox chemistry was also utilized to form polymer-coated
metal nanoparticles and mussel-inspired silver-releasing
antibacterial hydrogels [27]. Although this approach has been
successfully used for various important applications, one major
challenge remains: the ability to present a high density of
catechol functional groups in a defined ultrastructural
organization and architecture at the nano-scale.
[0019] There exists, therefore, an unmet need for bioadhesive,
anti-oxidant and/or antibacterial agents having a well-ordered
structures with highly-oriented functional groups.
SUMMARY OF THE INVENTION
[0020] The present invention provides self-assembled micro- and
nano-structures, having an ordered structure with controllable
orientation of sites that possess at least one of adhesive,
anti-bacterial, anti-fouling and/or anti-oxidant properties, or any
combination thereof. The micro- and nano-structures of the present
invention provide superior adhesive, anti-bacterial anti-fouling
and/or anti-oxidant properties as compared to currently known
products, and they are biocompatible, thus finding utility in a
variety of pharmaceutical, cosmetic and medical devices
applications.
[0021] The present invention is based in part on the concept of
mimicking adhesive, anti-oxidant anti-fouling and/or anti-bacterial
biological systems by incorporating DOPA functional groups in
self-assembling amino acids or peptides, with the aim of harnessing
the molecular self-assembly process to form well-ordered structures
endowed with functional properties due to a dense display of the
catecholic moieties. The ability of the aromatic amino acids or
short aromatic peptides to self-assemble into ordered
nanostructures, and the organization of end-capping groups such as
9-fluorenylmethoxycarbonyl (Fmoc)-modified amino acids/peptides
into hydrogels of nano-scale order allowed for the design of short
self-assembling building blocks containing DOPA and DOPA
derivatives.
[0022] Thus, the current invention employs molecular self-assembly
for generating micro-structures and nano-structures. More
specifically, the present invention is based on the unexpected
discovery that use of amino acids comprising catecholic moieties,
or incorporation of such amino acids into self-assembled peptides
provides adhesive, anti-microbial, anti-fouling and/or anti-oxidant
function to the resulting, self-assembled micro- or nano-structure,
and allows for the generation of a highly structured product with
superior properties. The well-ordered micro- or nano-structure of
said amino acids or peptides allows controlled orientation of
active moieties relative to the target surface, enhancing the
adhesive, anti-fouling, anti-microbial and/or anti-oxidant
properties of the product. Spatial orientation of the catecholic
moieties of the well-ordered self-assembled fibrillar micro- and
nano-structures of the present invention is schematically depicted
in FIG. 1. The depicted micro- and nano-structures provide a
surface comprised of the controllably exposed catecholic
groups.
[0023] One possible route of incorporating amino acids comprising
catecholic moieties is generating peptides comprising such amino
acids along with self-assembling protein motifs, such as, but not
limited to, the aromatic core recognition motif of the
.beta.-amyloid polypeptide--the di-phenylalanine dipeptide. The
diphenylalanine module efficiently self-assembles into discrete
well-ordered nanotubes [14]. These aromatic dipeptide nanotubes
(ADNT) are formed under mild conditions and possess high mechanical
stability and strength. In addition, ADNT can be aligned in a
controlled fashion both vertically and horizontally [15-17]. As
contemplated herein, by conjugating an amino acid comprising a
catecholic moiety (e.g., DOPA or a DOPA derivative) to the
di-phenylalanine motif and its derivatives, or by substituting the
phenylalanine moieties with one or more DOPA or DOPA derivative
moieties, well-ordered nanotubes may be created, wherein the DOPA
motif is displayed on the external wall of the tube. These
nanotubes may further be aligned to provide larger ordered
functional surface area. As demonstrated herein, the inventors have
found that addition of at least one amino acid comprising a
catecholic moiety to the diphenylalanine module, or substitution of
the diphenylalanine module with at least one amino acid comprising
a catecholic moiety (e.g., DOPA or a DOPA derivative), yielded
self-assembled micro- and nano-structures having at least one of
adhesive, anti-bacterial, anti-fouling and/or anti-oxidant
properties.
[0024] One currently preferred embodiment of the present invention
comprises substitution of one or more phenylalanine moieties with
amino acids comprising catecholic moieties (e.g., DOPA or a DOPA
derivative) in known peptide recognition motifs. As further
demonstrated herein, it has been shown that substituting aromatic
units in known peptide recognition motifs with amino acids
comprising catecholic moieties, yields self-assembled micro- and
nano-structures having at least one of adhesive, anti-bacterial,
anti-fouling and/or anti-oxidant properties.
[0025] Alternatively, it was unexpectedly discovered that
self-assembled bioadhesive micro-structure or nano-structure can be
formed from single amino acids comprising a catecholic moiety
(e.g., DOPA).
[0026] Thus, according to a first aspect, the present invention
provides a self-assembled micro- or nano-structure comprising (i) a
plurality of aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA) and a DOPA-derivative; or (ii)
a plurality of peptides, each peptide comprising between 2 and 9
amino acids, at least one of which is an aromatic amino acid
selected from 3,4-dihydroxyphenyl-L-alanine (DOPA) and a
DOPA-derivative; or (iii) a combination of said amino acids and
peptides; wherein said micro- or nano-structure has at least one
property selected from bioadhesive, anti-oxidant, anti-fouling,
anti-bacterial and any combination thereof. Each possibility
represents a separate embodiment of the present invention.
[0027] According to some embodiments, the micro- or nano-structure
is selected from the group consisting of a fibrillar
microstructure/nanostructure, a tubular
microstructure/nanostructure, a spherical
microstructure/nanostructure and a ribbon-like
microstructure/nanostructure. In one embodiment, the micro- or
nano-structure does not exceed about 500 nm in diameter. In another
embodiment, the micro- or nano-structure is at least about 1 nm in
diameter. Each possibility represents a separate embodiment of the
present invention.
[0028] According to some embodiments, each peptide in the plurality
of peptides comprises between 2 and 7 amino acids. Currently
preferred peptides comprise two amino acids (dipeptides), three
amino acids (tripeptides) or five amino acids (pentapeptides). Each
possibility represents a separate embodiment of the present
invention.
[0029] According to some embodiments, each peptide in the plurality
of peptides comprises a plurality of aromatic amino acids selected
from 3,4-dihydroxyphenyl-L-alanine (DOPA), a DOPA-derivative and a
combination thereof.
[0030] According to further embodiments, at least one peptide in
the plurality of peptides is a
3,4-dihydroxyphenyl-L-alanine-(3,4-dihydroxyphenyl-L-alanine)
(DOPA-DOPA) homodipeptide. According to further embodiments, the
DOPA-DOPA homodipeptide can be a dipeptide per se, or it can be
incorporated into the backbone of a longer peptide. Thus, according
to some embodiments, at least one peptide in the plurality of
peptides incorporates at least one
3,4-dihydroxyphenyl-L-alanine-(3,4-dihydroxyphenyl-L-alanine)
(DOPA-DOPA) homodipeptide in the peptide backbone. Optionally, the
DOPA-DOPA homopeptide further comprises at least one end-capping
modified moiety at the C- or N-terminus, as defined herein, for
example an Fmoc moiety.
[0031] In other embodiments, single amino acids (i.e., DOPA or
Fmoc-DOPA) can also self-assemble into micro- or nano-structures.
Accordingly, another embodiment of the present invention is
directed to a self-assembled bioadhesive micro- or nano-structure
comprising a plurality of aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA) and a DOPA-derivative.
[0032] In some embodiments, the present invention is directed to a
self-assembled micro- or nano-structure comprising a combination of
a plurality of single amino acids and a plurality of peptides, as
described herein.
[0033] In some embodiments, at least one amino acid or peptide in
the plurality of amino acids or peptides, preferably each amino
acid or peptide in the plurality of amino acids or peptides,
further comprises at least one amino acid capable of enhancing
cohesion, enhancing adhesion of said peptide to a surface, or a
combination thereof, thus rendering a bioadhesive micro- or
nano-structure. Preferably, the amino acid is charged at neutral
pH. In some embodiments, the amino acid comprises a positively
charged side chain capable of ionically interacting with negatively
charged surface, or a negatively charged side chain capable of
ionically interacting with positively charged surface. In currently
preferred embodiments, the amino acid is selected from the group
consisting of lysine, lysine analogs (e.g., ornithine), arginine,
aspartic acid, glutamic acid, and histidine.
[0034] A currently preferred amino acid for incorporation into the
plurality of DOPA containing peptides is lysine. As demonstrated
herein, it was unexpectedly found that incorporation of a lysine
residue into the DOPA-containing peptide, or conjugating lysine to
DOPA assemblies provides self-assembled structures with bioadhesive
properties. Without wishing to be bound by any particular theory or
mechanism of action, it is hypothesized that the incorporation of a
lysine residue into the DOPA-containing amino acid/peptide
assemblies contributes to cohesion and thus indirectly improve
adhesion. Moreover, lysine residues may also contribute to adhesion
via ionic bonding to negatively charged surfaces. Indeed, recent
evidence suggested that oxidation of DOPA residues to DOPA-quinone
or DOPA-semiquinone can lead to intermolecular cross-linking of the
MAPs, either with other DOPA residues by a radical mechanism or
with .epsilon.-amino group of lysine (Lys) residues [18]. Moreover,
lysine is a common residue in MAPs and the DOPA-lysine motif was
previously fused for the design of adhesive polymers [24].
[0035] According to some embodiments, the micro- or nano-structure
of the present invention further comprises at least one additional
amino acid, selected from the group consisting of naturally
occurring amino acids, synthetic amino acids and combinations
thereof. Each possibility represents a separate embodiment of the
present invention.
[0036] According to some embodiments, at least one amino acid or
peptide in the plurality of amino acids or peptides comprises at
least one end-capping modified moiety at the C- or N-terminus, or a
combination thereof. According to further embodiments, the end
capping moiety is selected from the group consisting of an aromatic
end capping moiety and a non-aromatic end-capping moiety. According
to still further embodiments, the end-capping moiety comprises a
labeling moiety.
[0037] According to some embodiments, aromatic end capping moiety
is selected from the group consisting of
9-fluorenylmethyloxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz).
According to additional embodiments, the non-aromatic end capping
moiety is selected from the group consisting of acetyl and
tert-butoxycarbonyl (Boc). Each possibility represents a separate
embodiment of the present invention. Additional examples of
end-capping amino acids include, but are not limited to,
naphthalene (Nap) derivatives, phenothiazine (PTZ)], azobenzene
(Azo), pyrene (Pyr), or cinnamoyl.
[0038] According to a certain embodiment, at least one of the
plurality of amino acids or peptides is selected from the group
consisting of Fmoc-DOPA, DOPA-DOPA, DOPA-Phe-Phe, Fmoc-DOPA-DOPA,
Fmoc-DOPA-DOPA-Lys, Fmoc-Phe-Phe-DOPA-DOPA-Lys,
Lys-Leu-Val-DOPA-DOPA-Ala-Glu, and Asp-DOPA-Asn-Lys-DOPA, as well
as and derivatives of any of the foregoing comprising an end
capping moiety, preferably an Fmoc moiety. Each possibility
represents a separate embodiment of the present invention.
[0039] According to some embodiments, the micro- or nano-structure
of the present invention is provided in the form of a hydrogel.
According to further embodiments, the hydrogel is characterized by
a storage modulus G1 ranging from .about.20 Pa to .about.5 kPa
according to the final concentration of the peptide, at 1 Hz
frequency, 0.7% strain.
[0040] According to additional embodiments, there is provided a
method of generating the self-assembled micro- or nano-structure
described herein, the method comprising the step of incubating a
plurality of amino acids or peptides under conditions which favor
formation of the micro- or nano-structure.
[0041] According to some embodiments, the micro- or nano-structure
of the present invention is capable of reducing a metal ion to
neutral metal atom, wherein the metal may be selected from the
group consisting of silver, gold, copper, platinum, nickel and
palladium. Each possibility represents a separate embodiment of the
present invention.
[0042] According to some embodiments, the micro- or nano-structure
of the present invention may be used in preparation of a
pharmaceutical composition, a cosmetic composition, or a medical
device (e.g., a medical sealant or adhesive such as an adhesive
patch or band-aid). In other embodiments, the micro- and
nano-structures of the present invention are applied as a coating
(e.g., an adhesive coating) to an existing medical device. Other
utilities include, but are not limited to a drug delivery vehicle,
a 3D scaffold for cell growth, tissue adhesive for regenerative
medicine, biological glue that is resilient to the shear forces of
blood flow, anti-bacterial and anti-oxidant uses, or any
combination of the foregoing.
[0043] According to a certain embodiment, the micro- or
nano-structure of the present invention may be used in the
preparation of biological glue.
[0044] According to other embodiments, the micro- or nano-structure
of the present invention may be used in the preparation of a
composition for combating bacteria or treating bacterial
infections.
[0045] According to other embodiments, the micro- or nano-structure
of the present invention may be used as an anti-oxidant, a radical
trapper, a metal chelator, or an oxidizable reducing agent.
[0046] In some embodiments, the micro- or nano-structures of the
present invention can be co-assembled with other self-assembled
peptides that are known in the art, such as Fmoc-Phe-Phe
Boc-Phe-Phe and Phe-Phe, or co-assembled with polypeptides,
polysaccharides, polymers, or a combination thereof.
[0047] In some embodiments, the micro- or nano-structures of the
present invention can be co-assembled with polysaccharides that are
known in the art to form hydrogels. Non-limiting examples of such
peptides are hyaluronic acid.
[0048] According to alternative embodiments, there is provided a
pharmaceutical composition, a cosmetic composition, a medical
device or a medical device coating, comprising the self-assembled
bioadhesive micro- or nano-structure of the present invention. The
pharmaceutical or cosmetic composition, or the device, may further
comprise a pharmaceutically or cosmetically acceptable carrier and
one or more additional excipients, which may vary depending on the
nature of the composition or device.
[0049] According to additional embodiments, there is provided a kit
for forming the self-assembled bioadhesive micro- or nano-structure
of the present invention, the kit comprising (i) a plurality of
aromatic amino acids selected from 3,4-dihydroxyphenyl-L-alanine
(DOPA) and a DOPA-derivative; or a plurality of peptides, each
peptide comprising between 2 and 9 amino acids, at least one of
which is an aromatic amino acid selected from
3,4-dihydroxyphenyl-L-alanine (DOPA) and a DOPA-derivative; or a
combination of said amino acids and peptides; and (ii) an aqueous
solution, each being individually packaged within the kit, wherein
the plurality of amino acids or peptides and the solution are
selected such that upon contacting said plurality of peptides and
said solution, said micro- or nano-structure is formed.
[0050] In other embodiments, the present invention relates to the
use of micro- or nano-structure described herein, for the
encapsulation of an agent selected from the group consisting of a
therapeutically active agent, a diagnostic agent, a biological
substance and a labeling moiety.
[0051] In other embodiments, the present invention relates to a
composition comprising the micro- or nano-structure as described
herein, and an agent selected from the group consisting of a
therapeutically active agent, a diagnostic agent, a biological
substance and a labeling moiety. The micro- or nano-structure may
in some embodiments encapsulate the agent, or in other embodiments
may be attached to said agent by any covalent or non-covalent
interactions. The agent may be selected from the group consisting
of therapeutically active agents, diagnostic agents, biological
substances and labeling moieties, such as, but not limited to
drugs, cells, proteins, enzymes, hormones, growth factors, nucleic
acids, organisms such as bacteria, fluorescence compounds or
moieties, phosphorescence compounds or moieties, and radioactive
compounds or moieties.
[0052] Further embodiments and the full scope of applicability of
the present invention will become apparent from the detailed
description given hereinafter. However, it should be understood
that the detailed description and specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1: Schematic representation of the self-assembled
bioadhesive nanostructures, comprising catecholic moieties.
[0054] FIG. 2A-2B: DOPA containing self-assembling peptides form
ordered ultrastructures. (FIG. 2A) Chemical structure of the
DOPA-containing designed peptides. (FIGS. 2B-2C) TEM micrographs of
DOPA-DOPA dipeptide assemblies; (FIGS. 2D-2F) TEM micrographs of
the hydrogel-forming Fmoc-DOPA-DOPA assemblies; (FIG. 2G) E-SEM
micrographs of the Fmoc-DOPA-DOPA hydrogel after gradual
dehydration.
[0055] FIGS. 3A-3F: Morphology characterization of Fmoc-DOPA-DOPA.
FIGS. 3A-3B TEM and FIGS. 3C-3D: HR-SEM images of Fmoc-DOPA-DOPA,
taken 24 hours after assembly, exhibiting tangled fibrous
structures. FIGS. 3E-3F: HR-SEM images taken 2 minutes after the
assembly, exhibiting large aggregates. Scale bar for the images is:
3A, 3C: 1 .mu.m; 3B: 200 nm; 3D: 100 nm; 3E, 3F: 10 .mu.m.
[0056] FIGS. 4A-4F: Rheological and structural properties of the
Fmoc-DOPA-DOPA hydrogelator. Strain sweep (FIG. 4A) and frequency
sweep (FIG. 4B) characterization of 5 mgmL.sup.-1 in situ-formed
hydrogel at 25.degree. C.; (FIG. 4C) Gelation kinetics of
Fmoc-DOPA-DOPA at different concentrations at 25.degree. C.; (FIG.
4D) Gelation kinetics of 5 mgmL.sup.-1 Fmoc-DOPA-DOPA at different
temperatures; (FIG. 4E) Kinetics of absorbance at 405 nm at two
concentrations and macroscopic visualization of the preparation;
(FIG. 4F) HR-SEM micrographs of the turbid peptide solution
immediately after inducing the assembly process (left and center
panels) and of the semi-transparent gel after 2 h of incubation
(right panel).
[0057] FIGS. 5A-5D: AFM measurements of Fmoc-DOPA-DOPA.
Measurements were conducted on 2 mg/ml Fmoc-DOPA-DOPA (FIG. 5A) or
5 mg/ml Fmoc-DOPA-DOPA (FIG. 5B). Samples were imaged using tapping
mode (40.times.40) and force/distance curves were determined at
several points (three repeats at each point). The measured adhesive
forces between the tip and the sample, represented by the minimum
value of the curves, are summarized in the tables (FIGS. 5C-5D) for
each of the samples.
[0058] FIGS. 6A-6B: Adhesion Force Map on Glass Slide (FIG. 6A).
Adhesion Histogram from Force Map (FIG. 6B), the mean force is
7.+-.1.2 nN.
[0059] FIGS. 7A-7C: AFM characterization of 5 mg/ml Fmoc-DOPA-DOPA
prepared in 5% ethanol solution: (FIG. 7A) AFM image (tapping mode,
scan size of 3.times.3 .mu.m2) of the dried hydrogel reveal the
presence of bulk fibrous structures. AFM image was obtained using
ultra sharp MicroMasch AFM probe. (FIG. 7B) Adhesion force map and
the corresponding histogram (FIG. 7C) of the sample, reveal mean
force of 36.+-.11 nN. Force data was obtained using SiO2 colloidal
probe. Tip velocity 1000 nm/s. Compressive force 20 nN.
[0060] FIGS. 8A-8C: Silver reduction by pre-prepared Fmoc-DOPA-DOPA
hydrogel. (FIG. 8A) Macroscopic visualization and UV-vis spectra of
assemblies at 5 mgmL-1 taken after five days of incubation; (FIG.
8B) TEM micrographs of the formation of silver particles after one
day of incubation of assemblies at 2.5 mgmL-1 (bottom panels) and a
control gel with no addition of silver nitrate (upper panel); (FIG.
8C) TEM micrographs of the assemblies at 5 mgmL-1 after two days of
incubation. The arrows indicate non-coated peptide assemblies. In
all micrographs, negative staining was not applied.
[0061] FIGS. 9A-9C: DOPA-DOPA reaction with silver nitrate. HR-SEM
images of 5 mg/ml DOPA-DOPA in the absence (FIG. 9A) or presence
(FIGS. 9B-9C) of silver nitrate. Silver deposition was observed in
the presence of silver nitrate. Scale bar for all images is 1
.mu.m.
[0062] FIGS. 10A-10B: DOPA-DOPA reaction with silver nitrate,
UV-visible extinction curves. Extinction values of 5 mg/ml
DOPA-DOPA at different pH in the presence or absence of silver
nitrate were measured between 250 to 800 nm, several hours after
silver nitrate was added to the solution (FIG. 10A). In the
presence of silver nitrate, a yellow color change resulting from a
peak centered at .about.410 nm occurred due to silver formation
(FIG. 10B--zoom-in).
[0063] FIGS. 11A-11D: AFM measurements of Fmoc-DOPA (5 mg/ml) at pH
5.5 Images of 5 mg/ml Fmoc-DOPA at pH 5.5 reveal sparse spheres at
varied sizes as shown by AFM (FIGS. 11A-11B) and light microscopy
(FIG. 11C). Force/distance curves were determined at several points
(three repeats at each point). The measured adhesive forces between
tip and sample, represented by the minimum value of the curves, are
summarized in a table (FIG. 11D).
[0064] FIGS. 12A-12G: AFM measurements of 5 mg/ml Fmoc-DOPA. The
experiment was conducted in water that were pre-adjusted to pH 8.7
by adding diluted NaOH solution. Images of Fmoc-DOPA (5 mg/ml)
reveal a dense layer of spheres at varied sizes. Small spheres are
observed at 3 .mu.m.times.3 .mu.m images (FIGS. 12A-12B), whereas
larger spheres are observed at the 40 .mu.m.times.40 .mu.m images
(FIGS. 12C-12D). Light microscopy image of the sample reveals a
dense layer of spheres (FIG. 12E). Force/distance curves were
determined at several points (three repeats at each point) for
Fmoc-DOPA (5 mg/ml) in the absence (FIG. 12F) or in the presence
(FIG. 12G) of Fe.sup.+3. The measured adhesive forces between tip
and sample, represented by the minimum value of the curves, are
summarized in a table for each of the samples.
[0065] FIGS. 13A-13D: AFM measurements of Fmoc-DOPA (2 mg/ml). The
experiment was conducted in water, and the pH was pre-adjusted to
pH 8.7 by adding diluted NaOH solution. Images of Fmoc-DOPA (2
mg/ml) reveal sparse spheres at varied sizes, as shown by AFM
(FIGS. 13A-13B) and light microscopy (FIG. 13C). The 3D image of
the sample indicates that the large spheres observed are truncated.
Force/distance curves were determined at several points (three
repeats at each point). The measured adhesive forces between tip
and sample, represented by the minimum value of the curves, are
summarized in a table (FIG. 13D).
[0066] FIGS. 14A-14C: AFM characterization of 1 mg/ml Fmoc-DOPA
prepared in 1% ethanol solution, after one day of assembly: (FIG.
14A) AFM image (tapping mode, scan size of 10 .mu.m.times.10 .mu.m)
reveal the presence of long fibers. AFM image was obtained using
ultra sharp MicroMasch AFM probe. (FIG. 14B) Adhesion force map and
the corresponding histogram (FIG. 14C) of the sample, reveal mean
force of 31.+-.10.6 nN. Tip velocity 1000 nm/s. Compressive force
20 nN.
[0067] FIGS. 15A-15E: Characterization of Fmoc-DOPA-DOPA-Lys
assemblies. (FIG. 15A) Chemical structure and TEM analysis of 1.25
wt % Fmoc-DOPA-DOPA-Lys assemblies prepared in either 12.5% ethanol
or 12.5% DMSO; (FIG. 15B) Adhesion force map and corresponding
histogram of 1.25% wt Fmoc-DOPA-DOPA-Lys prepared in ethanol and
water; (FIG. 15C) Adhesion force map and corresponding histogram of
1.25% wt Fmoc-DOPA-DOPA-Lys prepared in DMSO and water; (FIG. 15D)
AFM images of the exposed area of the bottom (left and center
panels) and top (right panel) glass surfaces after peeling two
glass slides that were adhered overnight by an aliquot of 1.25% wt
Fmoc-DOPA-DOPA-Lys in 12.5% ethanol; (FIG. 15E) AFM images of the
exposed area of the bottom (left and center panels) and top (right
panel) glass surfaces after peeling two glass slides that were
adhered overnight by a preparation of 1.25% wt Fmoc-DOPA-DOPA-Lys
in 12.5% DMSO.
[0068] FIGS. 16A-16F: Morphology characterization of DOPA-Phe-Phe
at low HFIP concentrations. TEM images of DOPA-Phe-Phe (2 mg/ml)
(FIGS. 16A-16C) or DOPA-Phe-Phe (5 mg/ml) (FIGS. 16D-16F) display
sphere-like structures. Scale bars for the images are: 16a: 100 nm;
16b: 200 nm; 16c: 1 .mu.m; 16d: 100 nm; 16e: 100 nm; 16f: 200
nm.
[0069] FIGS. 17A-17G: Morphology characterization of horizontally
aligned DOPA-Phe-Phe. SEM (FIGS. 17A-17B) and HR-SEM (FIGS.
17C-17G) images of the horizontally aligned peptide ribbon-like
structures. DOPA-Phe-Phe 50 mg/ml (FIGS. 17A-17B) or 100 mg/ml
(FIGS. 17A-17G), were dissolved in 100% HFIP and deposited on a
glass slide allowing rapid evaporation of the solvent, leading to
structure alignment. Scale bar for the images is: 17A: 20 .mu.m;
17B: 5 .mu.m; 17C: 1 .mu.m; 17D: 1 .mu.m; 17E: 1 .mu.m; 17F: 100
nm; 17G: 100 nm.
[0070] FIGS. 18A-18D: AFM measurements of 100 mg/ml DOPA-Phe-Phe.
Images of 100 mg/ml DOPA-Phe-Phe dissolved in 100% HFIP reveal
crowded arrangements of vertically aligned structures due to vapor
deposition, as shown by AFM (FIGS. 18A-18B) and light microscopy
(FIG. 18C). Force/distance curves were determined at several points
(three repeats at each point). The measured adhesive forces between
tip and sample, represented by the minimum value of the curves, are
summarized in a table (FIG. 18D).
[0071] FIGS. 19A-19F: AFM measurements of DOPA-Phe-Phe (50 mg/ml).
Images of 50 mg/ml DOPA-Phe-Phe dissolved in 100% HFIP reveal
disperse arrangements of structures, as shown by AFM (FIG. 19A) and
light microscopy (FIG. 19C). The 3D projection of the sample (FIG.
19B) reveals a wall-like structure, with cratered terrain (FIGS.
19D-19E). Force/distance curves were determined at several points
(three repeats at each point). The measured adhesive forces between
tip and sample, represented by the minimum value of the curves, are
summarized in a table (FIG. 19F).
[0072] FIGS. 20A-20B: Amino acid sequence of human calcitonin (hCT)
(SEQ ID. No. 1). Underlined are residues 15-19, which form the
minimal amyloidogenic recognition module of hCT; the chemical
structure of the module appears below (FIG. 20B). The chemical
structure of the hCT-inspired DOPA-containing pentapeptide,
Asp-DOPA-Asn-Lys-DOPA. The catechol hydroxyl substituents appear in
red (FIG. 20B).
[0073] FIGS. 21A-21C: High-resolution microscopy of fibrillar
assemblies formed by 6 mM Asp-DOPA-Asn-Lys-DOPA in water. (FIGS.
21A-21B) TEM micrographs, negative staining was applied. Scale bars
represent 2 .mu.m and 100 nm (FIG. 21C) E-SEM micrograph, scale bar
represents 1 .mu.m.
[0074] FIGS. 22A-22C: Congo Red (CR) staining of
Asp-DOPA-Asn-Lys-DOPA. 10 sample of 6 mM solution was stained with
CR and examined by (FIG. 22A) polarized optical microscopy and by
(FIG. 22B) fluorescence microscopy. Brightfield image corresponding
to the fluorescence microscopy micrograph. Scale bars represents
100 .mu.m (FIG. 22C).
[0075] FIGS. 23A-23B: Secondary structure analysis of
Asp-DOPA-Asn-Lys-DOPA. (FIG. 23A) FTIR spectrum of dried 6 mM
solution sample. The spectrum was analyzed by curve-fitting the
second derivative of the amide I' region. CD spectrum of 0.15 mM in
water at 25.degree. C. (FIG. 23B).
[0076] FIGS. 24A-24B: Temperature-dependent CD spectra of 0.15 mM
Asp-DOPA-Asn-Lys-DOPA in water (FIG. 24A). The temperature was
increased in a stepwise fashion from 18.degree. C. to 90.degree. C.
then similarly decreased to 18.degree. C. (FIG. 24B) Transmission
FTIR spectra of a dried sample that was taken from the CD cuvette
at the end of the experiment, a dried sample of the same solution
kept at room temperature and a baseline of water traces only.
Insets are respective TEM micrographs of the CD cuvette content at
the end of the experiment (FIG. 24B1 insert) and the solution kept
at room temperature (FIG. 24B2 insert). Negative staining was not
applied. Scale bars of the insets represent 200 nm.
[0077] FIGS. 25A-25C: TEM micrographs of 6 mM Asp-DOPA-Asn-Lys-DOPA
in water assembled at room temperature for four days. Solution
aliquot sampled after additional overnight incubation at room
temperature as control (FIG. 25A). Solution aliquot sampled after
additional overnight incubation at 37.degree. C. (FIG. 25B). The
same solution aliquot presented in the previous panel, sampled
after 8 h recovery at room temperature (FIG. 25C). For all samples,
negative staining was not applied. Scale bars represent 2
.mu.m.
[0078] FIGS. 26A-26B: Silver deposition on Asp-DOPA-Asn-Lys-DOPA
fibrillar assemblies following centrifugation, resuspension with
2.17 mM AgNO.sub.3 for 15 min and subsequent washing. TEM image
(FIG. 26A) of 15 mM peptide, negative staining was not applied.
Scale bar represents 200 nm (FIG. 26B) E-SEM image of 6 mM peptide.
Scale bar represents 5 .mu.m.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0079] The present invention provides self-assembled micro- and
nano-structures, having an ordered structure with controllable
orientation of adhesive, anti-microbial and/or anti-oxidant sites.
The micro- and nano-structures of the present invention provide
superior properties, e.g., at least one of adhesive and/or
anti-microbial and/or anti-oxidant and/or antifouling properties as
compared to currently known polymers, and they are biocompatible,
thus finding utility in a variety of pharmaceutical, cosmetic and
medical device applications.
[0080] Thus, according to one aspect of the present invention, the
self-assembled bioadhesive micro- and nano-structures is provided,
comprising a plurality of amino acids or peptides or a combination
thereof, wherein each amino acid is an aromatic amino acid
comprising a catecholic moiety, and/or wherein each peptide
comprises at least one aromatic amino acid comprising a catecholic
moiety. According to some embodiments, the at least one aromatic
amino acid is selected from the group consisting of:
3,4-dihydroxyphenyl-L-alanine (DOPA), a DOPA-derivative and a
combination thereof.
[0081] According to some aspects of the present invention,
self-assembled micro- and nano-structures is provided, comprising
(i) a plurality of aromatic amino acids selected from
3,4-dihydroxyphenyl-L-alanine (DOPA) and a DOPA-derivative; or (ii)
a plurality of peptides, each peptide comprising between 2 and 9
amino acids, at least one of which is an aromatic amino acid
selected from 3,4-dihydroxyphenyl-L-alanine (DOPA) and a
DOPA-derivative; or (iii) a combination of said amino acids and
peptides; wherein said micro- or nano-structure has at least one
property selected from bioadhesive, anti-oxidant, anti-fouling,
anti-bacterial and any combination thereof.
[0082] The term "DOPA derivative" as used herein refers to a
chemical derivative of DOPA including, but not limited to,
derivatization of any of the free functional groups of DOPA (i.e.,
carboxylic acid, amine or hydroxyl moieties). Examples of
carboxylic acid derivatives include amides (--CONH.sub.2) or esters
(--COOR), wherein R is alkyl, trihaloalkyl, alkenyl, alkynyl,
cycloalkyl, aryl or heteroaryl. Examples of amine functional groups
include, e.g., N-acylated derivatives (NH--COR), wherein R is
alkyl, trihaloalkyl, alkenyl, alkynyl, cycloalkyl, aryl or
heteroaryl. Examples of hydroxyl function group include O-acylated
derivatives (O--COR) or ether derivatives (OR) wherein R is alkyl,
trihaloalkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl.
Each possibility represents a separate embodiment of the present
invention. Derivatization of carboxylic acid moiety and/or amine
moiety refers to the embodiments where DOPA is in the terminal
position in the peptide (N-terminus or C-terminus).
[0083] According to some embodiments, at least one amino acid or
peptide in the plurality of amino acids or peptides in the micro-
or nano-structure of the present invention further comprises at
least one additional amino acid capable of enhancing cohesion,
enhancing adhesion of said peptide to a surface, or a combination
thereof, rendering a bioadhesive micro- or nano-structure.
Preferably, the amino acid is charged at neutral pH. In some
embodiments, the amino acid comprises a positively charged side
chain capable of ionically interacting with negatively charged
surface, or a negatively charged side chain capable of ionically
interacting with positively charged surface. In currently preferred
embodiments, the amino acid is selected from the group consisting
of lysine, lysine analogs (e.g., ornithine), arginine, aspartic
acid, glutamic acid, and histidine. A currently preferred amino
acid for incorporation into the plurality of DOPA-containing
peptides is lysine.
[0084] According to additional embodiments, the micro- or
nano-structure does not exceed about 50 .mu.m in diameter,
preferably does not exceed about 1 .mu.m in diameter. According to
further embodiments, the micro- or nano-structure does not exceed
about 500 nm in diameter. According to still further embodiments,
the micro- or nano-structure is at least 1 nm in diameter, e.g.,
about 1-50 nm, about 4-40 nm, about 10-30 nm, and the like. Each
possibility represents a separate embodiment of the present
invention.
[0085] In some embodiments, Fmoc-DOPA sphere diameter size range
between about 20 nm to several microns (no more then 5), and the
fiber width was less than about 20 nm. In other embodiments, the
Fmoc-DOPA-DOPA fiber width was about 4-30 nm. In other embodiments,
the DOPA-DOPA fiber width was about 20 to 50 nm Each possibility
represents a separate embodiment of the present invention.
[0086] In some embodiments, the micro- or nano-structures of the
present invention can be co-assembled with other self-assembled
peptides that are known in the art. Examples of such self-assembled
peptides are disclosed in U.S. Pat. No. 7,786,086, US 2009/0175785
and EP 1,575,867, the contents of each of which are incorporated by
reference herein. Examples of known self-assembled peptides that
can be combined or co-assembled with the DOPA-containing peptides
of the present invention are peptide-based hydrogels, composed of
short aromatic peptides (e.g., homodipeptides of aromatic amino
acid residues). The aromatic amino acid residues comprise, for
example, an aromatic moiety selected from the group consisting of
substituted or unsubstituted naphthalenyl, substituted or
unsubstituted phenanthrenyl, substituted or unsubstituted
anthracenyl, substituted or unsubstituted [1,10]phenanthrolinyl,
substituted or unsubstituted [2,2']bipyridinyl, substituted or
unsubstituted biphenyl and substituted or unsubstituted phenyl,
including, but not limited to: polyphenylalanine peptides,
polytriptophane peptides, and the like. Non-limiting examples
include phenylalanine-phenylalanine dipeptide,
naphthylalanine-naphthylalanine dipeptide,
phenanthrenylalanine-phenanthrenylalanine dipeptide,
anthracenylalanine-anthracenylalanine dipeptide,
[1,10]phenanthrolinylalanine-[1,10]phenanthrolinylalanine
dipeptide, [2,2']bipyridinylalanine-[2,2']bipyridinylalanine
dipeptide, (pentahalo-phenylalanine)-(pentahalo-phenylalanine)
dipeptide (including pentafluro phenylalanine, pentaiodo
phenylalanine, pentabromo phenylalanine, and pentachloro
phenylalanine dipeptides), phenylalanine-phenylalanine dipeptide,
(amino-phenylalanine)-(amino-phenylalanine) dipeptide,
(dialkylamino-phenylalanine)-(dialkylamino-phenylalanine)
dipeptide, (halophenylalanine)-(halophenylalanine) dipeptide,
(alkoxy-phenylalanine)-(alkoxy-phenylalanine) dipeptide,
(trihalomethyl-phenylalanine)-(trihalomethyl-phenylalanine)
dipeptide, (4-phenyl-phenylalanine)-(4-phenyl-phenylalanine)
dipeptide and (nitro-phenylalanine)-(nitro-phenylalanine)
dipeptide. Each of said peptides can further comprise an
end-capping moiety as described herein. Specific examples of
suitable peptides to be co-assembled with the peptides of the
present invention include Fmoc-Phe-Phe, Phe-Phe (wherein Phe is
phenylalanine), and halo-derivatives thereof, and the like.
[0087] According to some embodiments, the micro- or nano-structure
of the present invention further comprises at least one additional
amino acid, selected from the group consisting of naturally
occurring amino acids, synthetic amino acids and combinations
thereof. In other embodiments, all of the amino acids in the micro-
or nano-structures of the present invention comprise catecholic
moieties.
[0088] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0089] According to some embodiments, the micro- or nano-structure
of the present invention further comprises at least one naturally
occuring amino acid, selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic
acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine and
valine. Each possibility represents a separate embodiment of the
invention.
[0090] According to some embodiments, the micro- or nano-structure
of the present invention further comprises at least one
non-conventional or modified amino acid, selected from the group
consisting of halo-phenylalanine (including fluoro-phenylalanine,
bromo-phenylalanine, iodo-phenylalanine, chloro-phenylalanine,
pentafluro phenylalanine, pentaiodo phenylalanine, pentabromo
phenylalanine, and pentachloro phenylalanine), phenylglycine,
.alpha.-aminobutyric acid, .alpha.-amino-.alpha.-methylbutyrate,
aminocyclopropane-carboxylate, aminoisobutyric acid,
aminonorbornyl-carboxylate, cyclohexylalanine, cyclopentylalanine,
D-alanine, D-arginine, D-aspartic acid, D-cysteine, D-glutamine,
D-glutamic acid, D-histidine, D-isoleucine, D-leucine, D-lysine,
D-methionine, D-ornithine, D-phenylalanine, D-proline, D-serine,
D-threonine, D-tryptophan, D-tyrosine, D-valine,
D-.alpha.-methylalanine, D-.alpha.-methylarginine,
D-.alpha.-methylasparagine, D-.alpha.-methylaspartate,
D-.alpha.-methylcysteine, D-.alpha.-methylglutamine,
D-.alpha.-methylhistidine, D-.alpha.-methylisoleucine,
D-.alpha.-methylleucine, D-.alpha.-methyllysine,
D-.alpha.-methylmethionine, D-.alpha.-methylornithine,
D-.alpha.-methylphenylalanine, D-.alpha.-methylproline,
D-.alpha.-methylserine, D-.alpha.-methylthreonine,
D-.alpha.-methyltryptophan, D-.alpha.-methyltyrosine,
D-.alpha.-methylvaline, D-.alpha.-methylalnine,
D-.alpha.-methylarginine, D-.alpha.-methylasparagine,
D-.alpha.-methylasparatate, D-.alpha.-methylcysteine,
D-N-methylleucine, D-N-methyllysine, N-methylcyclohexylalanine,
D-N-methylornithine, N-methylglycine, N-methylaminoisobutyrate,
N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine,
N-(2-methylpropyl)glycine, D-N-methyltryptophan,
D-N-methyltyrosine, D-N-methylvaline, .gamma.-aminobutyric acid,
L-t-butylglycine, L-ethylglycine, L-homophenylalanine,
L-.alpha.-methylarginine, L-.alpha.-methylaspartate,
L-.alpha.-methylcysteine, L-.alpha.-methylglutamine,
L-.alpha.-methylhistidine, L-.alpha.-methylisoleucine,
D-N-methylglutamine, D-N-methylglutamate, D-N-methylhistidine,
D-N-methylisoleucine, D-N-methylleucine, D-N-methyllysine,
N-methylcyclohexylalanine, D-N-methylornithine, N-methylglycine,
N-methylaminoisobutyrate, N-(1-methylpropyl)glycine,
N-(2-methylpropyl)glycine, D-N-methyltryptophan,
D-N-methyltyrosine, D-N-methylvaline, .gamma.-aminobutyric acid,
L-t-butylglycine, L-ethylglycine, L-homophenylalanine,
L-.alpha.-methylarginine, L-.alpha.-methylaspartate,
L-.alpha.-methylcysteine, L-.alpha.-methylglutamine,
L-.alpha.-methylhistidine, L-.alpha.-methylisoleucine,
L-.alpha.-methylleucine, L-.alpha.-methylmethionine,
L-.alpha.-methylnorvaline, L-.alpha.-methylphenylalanine,
L-.alpha.-methylserine, L-.alpha.-methylvaline,
L-.alpha.-methylleucine,
N--(N-(2,2-diphenylethyl)carbamylmethyl-glycine,
1-carboxy-1-(2,2-diphenylethylamino)cyclopropane,
L-N-methylalanine, L-N-methylarginine, L-N-methylasparagine,
L-N-methylaspartic acid, L-N-methylcysteine, L-N-methylglutamine,
L-N-methylglutamic acid, L-N-methylhistidine,
L-N-methylisolleucine, L-N-methylleucine, L-N-methyllysine,
L-N-methylmethionine, L-N-methylnorleucine, L-N-methylnorvaline,
L-N-methylornithine, L-N-methylphenylalanine, L-N-methylproline,
L-N-methylserine, L-N-methylthreonine, L-N-methyltryptophan,
L-N-methyltyrosine, L-N-methylvaline, L-N-methylethylglycine,
L-N-methyl-t-butylglycine, L-norleucine, L-norvaline,
.alpha.-methyl-aminoisobutyrate,
.alpha.-methyl-.gamma.-aminobutyrate,
.alpha.-methylcyclohexylalanine, .alpha.-methylcyclopentylalanine,
.alpha.-methyl-.alpha.-napthylalanine, .alpha.-methylpenicillamine,
N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine,
N-(3-aminopropyl)glycine, N-amino-.alpha.-methylbutyrate,
.alpha.-napthylalanine, N-benzylglycine,
N-(2-carbamylethyl)glycine, N-(carbamylmethyl)glycine,
N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine,
N-cyclobutylglycine, N-cycloheptylglycine, N-cyclohexylglycine,
N-cyclodecylglycine, N-cyclododeclglycine, N-cyclooctylglycine,
N-cyclopropylglycine, N-cycloundecylglycine,
N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine,
N-(3-indolylyethyl)glycine, N-methyl-.gamma.-aminobutyrate,
D-N-methylmethionine, N-methylcyclopentylalanine,
D-N-methylphenylalanine, D-N-methylproline, D-N-methylserine,
D-N-methylserine, D-N-methylthreonine, N-(1-methylethyl)glycine,
N-methyla-napthylalanine, N-methylpenicillamine,
N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine,
L-.alpha.-methylalanine, L-.alpha.-methylasparagine,
L-.alpha.-methyl-t-butylglycine, L-methylethylglycine,
L-.alpha.-methylglutamate, L-.alpha.-methylhomophenylalanine,
N-(2-methylthioethyl)glycine, N-(3-guanidinopropyl)glycine,
N-(1-hydroxyethyl)glycine, N-(hydroxyethyl)glycine,
N-(imidazolylethyl)glycine, N-(3-indolylyethyl)glycine,
N-methyl-.gamma.-aminobutyrate, D-N-methylmethionine,
N-methylcyclopentylalanine, D-N-methylphenylalanine,
D-N-methylproline, D-N-methylserine, D-N-methylthreonine,
N-(1-methylethyl)glycine, N-methyla-napthylalanine,
N-methylpenicillamine, N-(p-hydroxyphenyl)glycine,
N-(thiomethyl)glycine, penicillamine, L-.alpha.-methylalanine,
L-.alpha.-methylasparagine, L-.alpha.-methyl-t-butylglycine,
L-methylethylglycine, L-.alpha.-methylglutamate,
L-.alpha.-methylhomophenylalanine, N-(2-methylthioethyl)glycine,
L-.alpha.-methyllysine, L-.alpha.-methylnorleucine,
L-.alpha.-methylornithine, L-.alpha.-methylproline,
L-.alpha.-methylthreonine, L-.alpha.-methyltyrosine,
L-N-methylhomophenylalanine, and
N--(N-(3,3-diphenylpropyl)carbamylmethyl(1)glycine. Each
possibility represents a separate embodiment of the invention.
[0091] The term "peptide" as used herein refers to a plurality of
amino acids (at least two), and encompasses native peptides
(including degradation products, synthetically synthesized peptides
or recombinant peptides) and peptidomimetics (typically,
synthetically synthesized peptides), as well as peptoids and
semipeptoids which are peptide analogs.
[0092] The term "amino acid comprising a catecholic moiety" refers
to, e.g., DOPA or a DOPA derivative as defined herein.
[0093] The micro- and nano-structures obtained by incorporating a
catecholic moiety comprising amino acid into the well-known
self-assembly peptide motifs were characterized and found to be in
various structural forms, such as, but not limited to, fibrilar,
tubular, spherical and ribbon-like structures, and any combination
thereof.
[0094] As used herein, the term "nano-structure" refers to a
physical structure, which in at least one dimension has a size
ranging from about 1 nm to less than about 1,000 nm, for example
about 10 nm or about 20 nm or about 50 nm to about 100 nm or about
200 nm or about 500 or less than about 1,000 nm.
[0095] As used herein, the term "micro-structure" refers to a
physical structure, which in at least one dimension has a size
ranging from about 1 .mu.m to about 100 .mu.m, for example about 10
.mu.m or about 20 .mu.m or about 50 .mu.m to about 100 .mu.m.
[0096] As used herein the phrase "tubular or spherical micro- or
nano-structure" refers to a spherical or elongated tubular or
conical structure having a diameter or a cross-section of less than
about 50 .mu.m (spherical structure) or less than about 500 nm
(tubular structure). The length of the tubular micro- or
nano-structure of the present invention is at least about 1 .mu.m.
It will be appreciated, though, that the tubular structure of the
present invention can be of infinite length (i.e., macroscopic
fibrous structures) and as such can be used in the fabrication of
hyper-strong materials.
[0097] As used herein the phrase "fibrillar nano-structure" refers
to a filament or fiber having a diameter or a cross-section of less
than about 100 nm. The length of the fibrillar nanostructure of the
present invention is preferably at least about 1 .mu.m. It will be
appreciated, though, that the fibrillar structure of the present
invention can be of infinite length (i.e., macroscopic fibrous
structures) and as such can be used in the fabrication of
hyper-strong materials.
[0098] As used herein the phrase "ribbon-like nano-structure"
refers to a filament or fiber, packed in a flat ribbon-like
structure, having a diameter or a cross-section of less than about
500 nm. The length of the ribbon-like nano-structure of the present
invention is preferably at least 1 about .mu.m. It will be
appreciated, though, that the ribbon-like structure of the present
invention can be of infinite length (i.e., macroscopic fibrous
structures) and as such can be used in the fabrication of
hyper-strong materials. Preferably, the ribbon-like nano-structures
described herein are characterized as non-hollowed or at least as
having a very fine hollow.
[0099] In some embodiments, the micro- or nano-structure of the
present invention is further characterized by adhesive properties.
Adhesion of the micro- and nano-structures to glass was measured by
atomic force microscopy (AFM), as described in the following
Examples. According to some embodiments, the shear strength of the
micro- and nano-structures on glass is from about 2 kPa to about 15
kPa.
[0100] In some embodiments of the present invention, the
self-assembled micro- or nano-structure may comprise a plurality of
amino acids comprising a catecholic moiety (e.g., DOPA or a DOPA
derivative such as Fmoc-DOPA). In other embodiments, the
self-assembled micro- or nano-structure may comprise a plurality of
peptides, each comprising between 2 and 9 amino acids. According to
a certain embodiment of this aspect of the present invention, each
peptide in said plurality of peptides comprises between 2 and 8
amino acids. According to a certain embodiment of this aspect of
the present invention, each peptide in said plurality of peptides
comprises between 2 and 7 amino acids. According to a certain
embodiment of this aspect of the present invention, each peptide in
said plurality of peptides comprises between 2 and 6 amino acids.
According to a certain embodiment of this aspect of the present
invention, each peptide in said plurality of peptides comprises
between 2 and 5 amino acids. According to a certain embodiment of
this aspect of the present invention, each peptide in said
plurality of peptides comprises between 2 and 4 amino acids.
According to a certain embodiment of this aspect of the present
invention, each peptide in said plurality of peptides comprises
between 2 and 3 amino acids. In currently preferred embodiments, at
least one peptide comprises two amino acids (dipeptide), three
amino acids (tripeptides) or five amino acids (pentapeptide). In
each of the aforementioned peptides, at least one amino acid
comprising a catecholic moiety (e.g., DOPA or a DOPA derivative
such as Fmoc-DOPA) is present.
[0101] In some embodiments, amino acid comprising a catecholic
moiety is located within the peptide sequence, such as, but not
limited to, Asp-DOPA-Asn-Lys-DOPA, Lys-Leu-Val-DOPA-DOPA-Ala-Glu,
Fmoc-Phe-Phe-DOPA-DOPA-Lys, DOPA-DOPA-Lys, and derivatives thereof
further comprising an end-capping moiety, for example Fmoc.
[0102] In other embodiments, the amino acids or peptides are
incorporated into a hybrid structure comprising the amino acids or
peptides comprising a catecholic moiety, in combination with other
amino acids or peptides in varying molar ratios. Non-limiting
examples of such hybrid structures include Fmoc-DOPA-DOPA+Fmoc-Lys;
Fmoc-DOPA-DOPA+Fmoc-Phe-Phe; Fmoc-DOPA-DOPA+Lys; and
Fmoc-DOPA-DOPA+DOPA. Each possibility represents a separate
embodiment of the present invention.
[0103] According to some embodiments, at least one of the peptides
in the plurality of peptides is a dipeptide. According to further
embodiments, the dipeptide is a homodipeptide. According to a
preferred embodiment, the homodipeptide is DOPA-DOPA dipeptide,
which may be a homodipeptide per se, or may be incorporated into a
longer peptide backbone. In accordance with this embodiment, a
plurality of DOPA-DOPA homodipeptides surprisingly formed tangled
fibril-like structure, in contrast to Phe-Phe homodipeptides, which
typically self-assemble into tubular structures.
[0104] According to further embodiments, at least one of the
peptides in said plurality of peptides is a tripeptide. According
to further embodiments, the tripeptide incorporates a homodipeptide
in the tripeptide backbone. According to some embodiments, the
tripeptide incorporates a DOPA-DOPA homopeptide in the backbone
thereof.
[0105] According to other embodiments, the peptides which form the
micro- and nano-structures of the present invention further
incorporate at least one aromatic amino acid comprising substituted
or unsubstituted naphthalenyl and substituted or -, halo, nitro,
azo, hydroxy, alkoxy, thiohydroxy, thioalkoxy, cyano, and
amine.
[0106] In one preferred embodiment, the present invention includes
the use of tripeptides including one aromatic amino acid comprising
a catecholic moiety, and a homodipeptide comprising aromatic
moieties.
[0107] Some non-limiting examples of homodipeptides, which can be
incorporated in the peptide backbone together with an aromatic
amino acid comprising a catechol moiety (e.g., DOPA), include
phenylalanine-phenylalanine,
(amino-phenylalanine)-(amino-phenylalanine),
(dialkylamino-phenylalanine)-(dialkylamino-phenylalanine),
halophenylalanine-halophenylalanine,
(alkoxy-phenylalanine)-(alkoxy-phenylalanine),
(trihalomethyl-phenylalanine)-(trihalomethyl-phenylalanine),
(4-phenyl-phenylalanine)-(4-phenyl-phenylalanine),
(nitro-phenylalanine)-(nitro-phenylalanine),
naphthylalanine-naphthylalanine,
anthracenylalanine-anthracenylalanine,
[1,10]phenanthrolinylalanine,-[1,10]phenanthrolinylalanine,
[2,2']bipyridinylalanine-[2,2']bipyridinylalanine, (4-phenyl
phenylalanine)-(4-phenyl phenylalanine) and
(p-nitro-phenylalanine)-(p-nitro-phenylalanine). According to the
preferred embodiment, the tripeptide DOPA-Phe-Phe is used.
[0108] The micro- and nano-structures of the present invention,
comprising an aromatic homodipeptide, such as Phe-Phe and an
aromatic amino acid comprising a catecholic moiety, were
surprisingly found to self-assemble into sphere particles or
ribbon-like micro- and nano-structures, in contrast to Phe-Phe
homodipeptides, which typically self-assemble into tubular
structures.
[0109] The present invention also encompasses micro- and
nano-structures comprising a plurality of longer peptides, wherein
at least one of the plurality of peptides comprises 4-9 amino
acids, preferably 4-7 amino acids. According to some embodiments,
each of the plurality of peptides comprises 4-9 amino acids,
preferably 4-7 amino acids. Said peptides may be based on fragments
of amyloidogenic proteins that were shown to form typical
amyloid-like structures. The adhesive, anti-microbial, anti-oxidant
and/or anti-fouling properties of said micro- and nano-structures
are provided by substituting phenylalanine present in said
fragments by at least one amino acid comprising a catecholic moiety
(e.g., DOPA). According to some embodiments, said fragments further
comprise lysine residue, which is another main constituent of
mussel adhesive proteins. Lysine residue may contribute to adhesion
via ionic bonding to negatively charged surfaces, and
intermolecular cross-linking with o-quinones [18]. Some
non-limiting examples of such proteins are
Lys-Leu-Val-DOPA-DOPA-Ala-Glu and Asp-DOPA-Asn-Lys-DOPA.
Lys-Leu-Val-DOPA-DOPA-Ala-Glu is based on
Lys-Leu-Val-Phe-Phe-Ala-Glu heptapeptide fragment of the
.beta.-amyloid peptide associated with Alzheimer's disease
(A.beta.16-22) that was shown to form highly ordered amyloid
fibrils and tubular structures [19-20]. Asp-DOPA-Asn-Lys-DOPA is
based on Asp-Phe-Asn-Lys-Phe (SEQ ID. No. 2), a pentapeptide
fragment derived from the human calcitonin (hCT) polypeptide
hormone that was shown to form amyloid-like fibrils [15].
[0110] The present invention also envisages self-assembled
bioadhesive micro- and nano-structures which are composed of a
plurality of peptides being longer than the above described (e.g.,
10-150 amino acids), wherein each peptide comprises at least one
aromatic amino acid comprising a catecholic moiety (e.g.,
DOPA).
[0111] The micro- and nano-structures of the present invention may
further comprise end-capping modified amino acids or peptides.
Thus, according to an embodiment of the present invention, at least
one of the plurality of peptides of the self-assembled bioadhesive
micro- or nano-structure is modified by one or more aromatic end
capping moiety. According to another embodiment, each of the
plurality of peptides of the self-assembled bioadhesive micro- or
nano-structure is modified by one or more aromatic end capping
moiety. The peptides may further be modified by one or more
non-aromatic end capping moiety.
[0112] The phrase "end-capping modified moiety", as used herein,
refers to an amino acid or peptide which has been modified at the
N-(amine) terminus and/or the C-(carboxyl) terminus thereof. The
end-capping modification refers to the attachment of a chemical
moiety to the terminus, so as to form a cap. Such a chemical moiety
is referred to herein as an end capping moiety and is typically
also referred to herein and in the art, interchangeably, as a
peptide protecting moiety or group.
[0113] Representative examples of aromatic end capping moieties
suitable for N-terminus modification include, without limitation,
fluorenylmethyloxycarbonyl (Fmoc), naphthalene (Nap) derivatives,
phenothiazine (PTZ)], azobenzene (Azo), pyrene (Pyr), and
cinnamoyl.
[0114] Representative examples of non-aromatic end capping moieties
suitable for N-terminus modification include, without limitation,
formyl, acetyl trifluoroacetyl, tert-butoxycarbonyl (Boc),
trimethylsilyl, and 2-trimethylsilyl-ethanesulfonyl.
[0115] Representative examples of non-aromatic end capping moieties
suitable for C-terminus modification include, without limitation,
amides, allyloxycarbonyl, trialkylsilyl ethers and allyl ethers.
Representative examples of aromatic end capping moieties suitable
for C-terminus modification include benzyl, benzyloxycarbonyl
(Cbz), trityl and substituted trityl groups.
[0116] According to several embodiments, micro- and nano-structures
comprising end-capping modified amino acids or peptides include
Fmoc-DOPA, Fmoc-DOPA-DOPA and Fmoc-DOPA-DOPA-Lys. According to
further embodiments, the micro- and nano-structures comprising
end-capping modified amino acids or peptides include
Fmoc-Phe-Phe-DOPA-DOPA-Lys.
[0117] The end-capping modification changes the structure of the
end-capping of the peptide, changing its chemical and physical
properties and therefore changing the chemical and physical
properties of the peptide and the chemical and physical properties
of the resulting nanostructure. Using such end-capping
modification, micro- and nano-structures in which these properties
are finely controlled can be formed and hence, controlled
fabrication of e.g., films, monolayer, or other macroscopic
structures with nano-scale order is allowed.
[0118] As demonstrated in the following Examples section, it was
found that aromatic amino acids comprising a catecholic moiety, or
peptides comprising such amino acids, which are modified with an
aromatic end-capping moiety, self-assembles into sphere-like
particles or into a fibrillar micro- or nano-structures, having
adhesive properties. The formation of bioadhesive fibrillar micro-
and nano-structures was particularly observed while utilizing
DOPA-DOPA homodipeptides modified by an aromatic end-capping
moiety, similarly to Phe-Phe end-capping modified homodipeptides,
and the formation of bioadhesive spheres was observed while
utilizing Fmoc-DOPA building blocks.
[0119] As is further demonstrated and discussed in the Examples
section that follows, it was found that the fibrillar
nano-structure in addition to the adhesive property was
characterized by macroscopic properties of a hydrogel, with storage
modulus (G') ranging that could be modulated with high dynamic
range from .about.20 Pa to 5 kPa.
[0120] As the micro- and nano-structures of the present invention
comprise an aromatic amino acid having a catecholic moiety, wherein
a catechol group has redox properties, said micro- and
nano-structures are capable of reducing a metal ion to neutral
metal atom. This trait was previously utilized to form metal
core-polymer shell nanoparticles [21] and mussel-inspired
silver-releasing antibacterial hydrogels [22]. As demonstrated
herein, it was found that the hydrogels of the present invention
were capable of reducing silver nitrate to spontaneously form
silver particles, as described in following Examples. As the
reduction of metal ions is performed by the catechloic groups of
the plurality of peptides, which are controllably presented upon
the micro- or nano-structure, the orientation of metal
nanoparticles may be accordingly controlled. Thus, the
self-assembled micro- and nano-structures may comprise a metal,
wherein said metal is at least partially enclosed by a discrete
fibrillar micro- or nano-structure. The self-assembled micro- and
nano-structures may further comprise a metal controllably deposited
on the outer shell of the fibrillar nano-structure. The metals,
which can be deposited on the micro- and nano-structures include,
but are not limited to, silver, gold, copper, platinum, nickel and
palladium.
[0121] Based on the redox properties of the micro- or
nano-structures of the present invention, they are useful as an
ant-oxidant composition. Furthermore, the micro- or nano-structures
of the present invention may be used as a radical trapper, a metal
chelator, or an oxidizable reducing agent. Alternatively, the
micro- or nano-structures of the present invention may be used for
preparing compositions for combating bacteria or treating bacterial
infections. Each possibility represents a separate embodiment of
the present invention.
[0122] The term "anti-bacterial" may refer to one or more of the
following effects: killing the bacteria (bacteriocide), causing
halt of growth of bacteria (bacteriostatic), prevention of
bacterial infection, prevention of bio-film formation and
disintegration of a formed biofilm, and decrease in bacterial
virulence.
[0123] Examples of bacterial strain that can be treated/disinfected
by the composition of the invention (both as a disinfecting
composition and as a pharmaceutical composition) are all gram
negative and gram positive bacteria and in particular pathogenic
gram negative and gram positive bacteria.
[0124] The term "combating bacteria" or "treating bacterial
infection" may refer to one of the following: decrease in the
number of bacteria, killing or eliminating the bacteria, inhibition
of bacterial growth (stasis), inhibition of bacterial infestation,
inhibition of biofilm formation, disintegration of existing
biofilm, or decrease in bacterial virulence.
[0125] In other embodiments of the present invention, fibrous
network of the micro- and nano-structures, which have a form of a
hydrogel, may contain microscopic hollow cavities. This structural
feature indicates that such hydrogel can be utilized as a matrix
for encapsulating or attaching various agents thereto. In addition,
these hollow cavities further enable to entrap therein biological
substances such as cells (e.g., neural cells), allowing expansion
and elongation of the cells within the hydrogel.
[0126] Agents that can be beneficially encapsulated in or attached
to the hydrogel include, for example, therapeutically active
agents, diagnostic agents, biological substances and labeling
moieties. More particular examples include, but are not limited to,
drugs, cells, proteins, enzymes, hormones, growth factors, nucleic
acids, organisms such as bacteria, fluorescence compounds or
moieties, phosphorescence compounds or moieties, and radioactive
compounds or moieties.
[0127] As used herein, the phrase "therapeutically active agent"
describes a chemical substance, which exhibits a therapeutic
activity when administered to a subject. These include, as
non-limiting examples, inhibitors, ligands (e.g., receptor agonists
or antagonists), co-factors, anti-inflammatory drugs (steroidal and
non-steroidal), antipsychotic agents, analgesics, anti-thrombogenic
agents, anti-platelet agents, anticoagulants, anti-diabetics,
statins, toxins, antimicrobial agents, anti-histamines,
metabolites, anti-metabolic agents, vasoactive agents, vasodilator
agents, cardiovascular agents, chemotherapeutic agents,
antioxidants, phospholipids, anti-proliferative agents and
heparins.
[0128] As used herein, the phrase "biological substance" refers to
a substance that is present in or is derived from a living organism
or cell tissue. This phrase also encompasses the organisms, cells
and tissues. Representative examples therefore include, without
limitation, cells, amino acids, peptides, proteins,
oligonucleotides, nucleic acids, genes, hormones, growth factors,
enzymes, co-factors, antisenses, antibodies, antigens, vitamins,
immunoglobulins, cytokines, prostaglandins, vitamins, toxins and
the like, as well as organisms such as bacteria, viruses, fungi and
the like.
[0129] As used herein, the phrase "diagnostic agent" describes an
agent that upon administration exhibits a measurable feature that
corresponds to a certain medical condition. These include, for
example, labeling compounds or moieties, as is detailed
hereinunder. As used herein, the phrase "labeling compound or
moiety" describes a detectable moiety or a probe which can be
identified and traced by a detector using known techniques such as
spectral measurements (e.g., fluorescence, phosphorescence),
electron microscopy, X-ray diffraction and imaging, positron
emission tomography (PET), single photon emission computed
tomography (SPECT), magnetic resonance imaging (MRI), computed
tomography (CT) and the like.
[0130] Representative examples of labeling compounds or moieties
include, without limitation, chromophores, fluorescent compounds or
moieties, phosphorescent compounds or moieties, contrast agents,
radioactive agents, magnetic compounds or moieties (e.g.,
diamagnetic, paramagnetic and ferromagnetic materials), and heavy
metal clusters, as is further detailed hereinbelow, as well as any
other known detectable moieties.
[0131] As used herein, the term "chromophore" refers to a chemical
moiety or compound that when attached to a substance renders the
latter colored and thus visible when various spectrophotometric
measurements are applied. A heavy metal cluster can be, for
example, a cluster of gold atoms used, for example, for labeling in
electron microscopy or X-ray imaging techniques.
[0132] As used herein, the phrase "fluorescent compound or moiety"
refers to a compound or moiety that emits light at a specific
wavelength during exposure to radiation from an external source. As
used herein, the phrase "phosphorescent compound or moiety" refers
to a compound or moiety that emits light without appreciable heat
or external excitation, as occurs for example during the slow
oxidation of phosphorous.
[0133] As used herein, the phrase "radioactive compound or moiety"
encompasses any chemical compound or moiety that includes one or
more radioactive isotopes. A radioactive isotope is an element
which emits radiation. Examples include .alpha.-radiation emitters,
.beta.-radiation emitters or .gamma.-radiation emitters.
[0134] While a labeling moiety can be attached to the hydrogel, in
cases where the one or more of the peptides composing the hydrogel
is an end-capping modified peptide, the end-capping moiety can
serve as a labeling moiety per se.
[0135] Thus, for example, in cases where the Fmoc group described
hereinabove is used as the end-capping moiety, the end-capping
moiety itself is a fluorescent labeling moiety. In another example,
wherein the Fmoc described hereinabove further includes a
radioactive fluoro atom (e.g., .sup.18F) is used as the end-capping
moiety, the end-capping moiety itself is a radioactive labeling
moiety.
[0136] Other materials which may be encapsulated by the hydrogel of
the present invention include, without limitation, conducting
materials, semiconducting materials, thermoelectric materials,
magnetic materials, light-emitting materials, biominerals, polymers
and organic materials.
[0137] Each of the agents described herein can be attached to or
encapsulated in the hydrogel by means of chemical and/or physical
interactions. Thus, for example, compounds or moieties can be
attached to the external and/or internal surface of the hydrogel,
by interacting with functional groups present within the hydrogel
via, e.g., covalent bonds, electrostatic interactions, hydrogen
bonding, van der Waals interactions, donor-acceptor interactions,
aromatic (e.g., .pi.-.pi. interactions, cation-.pi. interactions
and metal-ligand interactions. These interactions lead to the
chemical attachment of the material to the peptide fibrous network
of the hydrogel. As an example, various agents can be attached to
the hydrogel via chemical interactions with the side chains,
N-terminus or C-terminus of the peptides composing the hydrogel
and/or with the end-capping moieties, if present.
[0138] Alternatively, various agents can be attached to the
hydrogel by physical interactions such as magnetic interactions,
surface adsorption, encapsulation, entrapment, entanglement and the
likes.
[0139] The micro- and nano-structures of the present invention are
preferably generated by allowing a highly concentrated aqueous
solution of the peptides of the present invention to self-assemble
under mild conditions as detailed in Example 1 of the Examples
section which follows.
[0140] Alternatively, the preparation of the hydrogel can also be
performed upon its application, such that the plurality of peptides
and the aqueous solution are each applied separately to the desired
site and the hydrogel is formed upon contacting the peptides and
the aqueous solution at the desired site of application. Thus, for
example, contacting the peptides and the aqueous solution can be
performed in vivo, such that the plurality of peptides and the
aqueous solution are separately administered.
[0141] According to these embodiments, the administration is
preferably effected locally, into a defined bodily cavity or organ,
where the plurality of peptides and the aqueous solution become in
contact while maintaining the desired ratio therebetween that would
allow the formation of a self-assembled bioadhesive nanostructure
within the organ or cavity.
[0142] Using such a route of preparing the hydrogel in vivo allows
to beneficially utilize the formed micro- or nano-structure in
applications such as, for example, dental procedures, as a dental
glue, dental implant or filling material, cosmetic applications,
tissue regeneration, implantation, and in would healing, as a wound
dressing that is formed at a bleeding site, as is further detailed
hereinbelow.
[0143] Thus, according to another aspect of the present invention
there is provided a kit for forming the micro- and nano-structures
described herein which comprise a plurality of amino acids or
peptides, as described herein and an aqueous solution, as described
herein, each being individually packaged within the kit, wherein
the plurality of peptides and the solution are selected such that
upon contacting the plurality of peptides and the solution, a
self-assembled bioadhesive nanostructure, as described herein, is
formed.
[0144] Such a kit can be utilized to prepare the micro- and
nano-structures described herein at any of the desired site of
actions (e.g., a bodily cavity or organ) described hereinabove. The
kit can be designed such that the plurality of specific peptides
and the aqueous solution would be in such a ratio that would allow
the formation of the desired micro- and nano-structure at the
desired site of application.
[0145] As used herein, the phrases "desired site of application"
and "desired application site" describe a site in which application
of the micro- and nano-structures of the present invention is
beneficial, namely, in which the micro- and nano-structures can be
beneficially utilized for therapeutic, diagnostic, cosmetic, and/or
biomedical applications, as described in detail hereinbelow. Such a
kit can further comprise an active agent, as is detailed
hereinbelow.
[0146] The active agent can be individually packaged within the kit
or can be packaged along with the plurality of peptides or along
with the aqueous solution.
[0147] By being remarkably adhesive and having a well-ordered
structure, and further by being stable, biocompatible and capable
of encapsulating therein of various agents, the micro- and
nano-structures described herein can be beneficially utilized in
various applications, as is detailed hereinunder.
[0148] The bioadhesive, anti-microbial and/or anti-oxidant
self-assembled micro- and nano-structures described herein can, for
example, form a part of pharmaceutical or cosmetic compositions,
either alone or in the presence of a pharmaceutically or
cosmetically acceptable carrier. The micro- and nano-structures
described herein may further be used in medical devices (e.g., a
medical sealant or adhesive). In other embodiments, the micro- and
nano-structures of the present invention are applied as a coating
(e.g., an adhesive coating) to an existing medical device.
[0149] As used herein, a "pharmaceutical or cosmetic composition"
refers to a preparation of the micro- and nano-structures described
herein, with other chemical components such as acceptable and
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism. The purpose of a cosmetic composition is typically to
facilitate the topical application of a compound to an organism,
while often further providing the preparation with aesthetical
properties. Hereinafter, the term "pharmaceutically, or
cosmetically acceptable carrier" refers to a carrier or a diluent
that does not cause significant irritation to an organism and does
not abrogate the biological activity and properties of the applied
compound. Examples, without limitations, of carriers include
propylene glycol, saline, emulsions and mixtures of organic
solvents with water, as well as solid (e.g., powdered) and gaseous
carriers.
[0150] The compositions described herein may be formulated in
conventional manner using one or more acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the nanoparticles into preparations. Proper formulation is
dependent upon the route of administration chosen. Techniques for
formulation and administration of drugs may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., latest edition.
[0151] The pharmaceutical compositions described herein can be
formulated for various routes of administration. Suitable routes of
administration may, for example, include oral, sublingual,
inhalation, rectal, transmucosal, transdermal, intracavemosal,
topical, intestinal or parenteral delivery, including
intramuscular, subcutaneous and intramedullary injections as well
as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, or intraocular injections. Bioadhesive
self-assembled compositions of the present invention are
particularly useful for transdermal drug delivery systems, as
bioadhesives incorporated into pharmaceutical formulations allow
enhancing the drug absorption by mucosal cells and provide
timed-release of the drug to the target mucosal site [23].
[0152] Formulations for topical administration include but are not
limited to lotions, ointments, gels, creams, suppositories, drops,
liquids, sprays and powders. Conventional carriers, aqueous, powder
or oily bases, thickeners and the like may be necessary or
desirable.
[0153] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
sachets, capsules or tablets. Thickeners, diluents, flavorings,
dispersing aids, emulsifiers or binders may be desirable.
Formulations for parenteral administration may include, but are not
limited to, sterile solutions which may also contain buffers,
diluents and other suitable additives. Timed oral compositions are
envisaged for treatment.
[0154] The micro- and nano-structures may be applied as a coating
to a solid oral dosage formulation, such as a tablet or gel-capsule
or to a transmucosal drug delivery device. The bioadhesive micro-
and nano-structure may further be present in the matrix of a tablet
or transmucosal drug delivery device. The micro- and
nano-structures of the present invention may further encapsulate
the drug or to function as a shell in the core-shell tablet or
device, wherein the core comprises a drug to be delivered.
[0155] The medical devices incorporating the micro- and
nano-structures of the present invention may include a biologic
glue, an implant, an artificial body part, a tissue engineering and
regeneration system, a wound dressing, a synthetic skin, a cell
culture matrix, a protein microarray chip, a biosensor, an
anastomotic device (e.g., stent), a sleeve, an adhesive film, a
scaffold and a coating. Bioadhesive micro- and nano-structures are
particularly useful in medical devices configured to provide
adhesion or requiring adhesive properties in order to function, for
example a biological glue, a wound dressing, a synthetic skin, an
adhesive film, or a coating. Use of the bioadhesive micro- and
nano-structures of the present invention as a biological glue is
particularly beneficial, as there is a significant unmet clinical
need for a strong and flexible surgical glue that is highly
biocompatible. Introducing adhesive properties to medical devices,
such as, but not limited to, an artificial body part, a tissue
engineering and regeneration system, a cell culture matrix, a
protein microarray chip, a biosensor, an anastomotic device, a
sleeve, or a scaffold, which do not generally require inherent
adhesion, may still be beneficial. For example, the adhesive
properties of said devices may eliminate a need in using additional
substances, such as glue, for permanently or releasably attaching
the device to the target surface.
[0156] In addition to being used as medical devices or being
incorporated into medical devices, the micro- and nano-structures
of the present invention may be used to provide adhesive coating
layers to existing medical devices, or for forming adhesive medical
devices, e.g., band-aids.
[0157] Sleeves comprising the micro- and nano-structures or
compositions described herein can be used, for example, as outside
scaffolds for nerves and tendon anastomoses (the surgical joining
of two organs).
[0158] Adhesive films comprising the micro- and nano-structures or
compositions described herein can be used, for example, as wound
dressing, substrates for cell culturing and as abdominal wall
surgical reinforcement.
[0159] Coatings of medical devices comprising the micro- and
nano-structures or compositions described herein can be used to
render the device biocompatible, having a therapeutic activity, a
diagnostic activity, and the like. Other devices include, for
example, catheters, aortic aneurysm graft devices, a heart valve,
indwelling arterial catheters, indwelling venous catheters,
needles, threads, tubes, vascular clips, vascular sheaths and drug
delivery ports.
[0160] The self-assembled micro- and nano-structures may further be
incorporated as cosmetic agents.
[0161] As used herein, the phrase "cosmetic agent" refers to
topical substances that are utilized for aesthetical purposes.
Cosmetic agents in which the micro- and nano-structures and
compositions described herein can be beneficially utilized include,
for example, agents for firming a defected skin or nail, make ups,
gels, lacquers, eye shadows, lip glosses, lipsticks, and the
like.
[0162] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. As used herein,
the singular form "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. For example, the
term "a compound" or "at least one compound" may include a
plurality of compounds, including mixtures thereof.
[0163] The foregoing examples of the related art and limitations
related therewith are intended to be illustrative and not
exclusive. Other limitations of the related art will become
apparent to those of skill in the art upon a reading of the
specification and a study of the figures.
[0164] All references cited herein are hereby incorporated by
references in their entirety herein.
EXAMPLES
Example 1
Experimental Methods
[0165] Nanostructures Self-Assembly
[0166] Material--Peptides (Fmoc-DOPA-DOPA, DOPA-DOPA, DOPA-Phe-Phe,
Fmoc-DOPA-DOPA-Lys) were purchased from Peptron. Fmoc-DOPA was
purchased from Ana Spec. A stock solution of Fmoc-DOPA was prepared
by dissolving the building block with ethanol to a final
concentration of 100 mg/ml. The stock solution was then diluted
into water to the desired concentration (0.5 mg/ml, 0.75 mg/ml, 1
mg/ml, or 2 mg/ml). DOPA-Phe-Phe was prepared by dissolving
lyophilized form of the peptide in
1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP), at a concentration of
100 mg/ml or 50 mg/ml. The stock solution was either directly
deposited on a cover slip glass slides or diluted into water to a
final concentration of 2 mg/ml or 5 mg/ml.
[0167] For the formation of the DOPA-DOPA dipeptide asssemblies
(FIG. 2A-left panel), lyophilized peptide was dissolved in ethanol
to a concentration of 33 mg/mL then diluted with Milli-Q water to a
final concentration of 5 mg/mL.
[0168] For the formation of the Fmoc-DOPA-DOPA dipeptide assemblies
(FIG. 2A-right panel), lyophilized peptide was dissolved in ethanol
to a concentration of 100 mg/mL then diluted with Milli-Q water to
the desired concentration (2.5 mg/mL, 5 mg/mL or 10 mg/mL).
[0169] For formation of Fmoc-DOPA-DOPA-Lys assemblies (FIG. 8A),
lyophilized peptide was dissolved in either ethanol or dimethyl
sulfoxide (DMSO) to a concentration of 100 mg/mL then diluted with
Milli-Q water to a final concentration of 12.5 mg/mL.
[0170] Asp-DOPA-Asn-Lys-DOPA pentapeptide was synthesized by
Peptron Inc. (Daejeon, Korea). To induce the formation of fibrillar
assemblies, lyophilized peptide was dissolved in ultra-pure water
to concentrations of 100 .mu.M to 15 mM by vortexing followed by
bath-sonication for 10 min To avoid pre-aggregation, fresh stock
solutions were prepared for each experiment.
[0171] Transmission Electron Microscopy (TEM)
[0172] TEM analysis was performed by applying 10 .mu.L samples to
400-mesh copper grids covered by carbon-stabilized Formvar film.
The samples were allowed to adsorb for 2 min before excess fluid
was blotted off. For samples that were negatively stained, 10 .mu.L
of 2% uranyl acetate were then deposited on the grid and allowed to
adsorb for 2 min before excess fluid was blotted off. TEM
micrographs were recorded using JEOL 1200EX electron microscope
(Tokyo, Japan) operating at 80 kV.
[0173] High Resolution Scanning Electron Microscopy (HR-SEM) for
Hydrogel Characterization
[0174] HR-SEM analysis was done for Fmoc-DOPA-DOPA hydrogels at
different time points after peptide self-assembly. 10 .mu.L samples
were dried at room temperature on microscope glass cover slips and
coated with chromium. Images were taken using a JEOL JSM 6700F
FE-SEM operating at 10 kV.
[0175] Environmental Scanning Electron Microscopy E-SEM
[0176] E-SEM analysis of 5 mg/mL Fmoc-DOPA-DOPA hydrogel was
performed by placing a portion of a pre-prepared hydrogel on a
microscope metal stand. Images were taken using Quanta 200 Field
Emission Gun (FEG) E-SEM (FEI, Eindhoven, the Netherlands)
operating at 10 kV.
Adhesion Measurements
Embodiment 1
[0177] Atomic Force Microscopy (AFM) was used to assess the
adhesion of the formed structures to a silicon oxide tip by
employing "force/distance" measurements. This type of measurements
allows deducing the attractive forces between the AFM tip and the
contacted surface, when this force is represented by the minimum
value of a force/distance curve. For AFM analysis, 10 .mu.l aliquot
of the peptide suspension was deposited on clean glass slide and
dried at room temperature. Force/distance curves were determined at
several points using NanoWizardIII of JPK instruments AG.
Adhesion Measurements
Embodiment 2
[0178] In an alternative embodiment, AFM analysis was performed
using an Asylum MFP-1D AFM instrument (Asylum Research, Santa
Barbara, Calif., USA). To obtain force data the different peptides
were prepared in ethanol: Fmoc-DOPA (1%) Fmoc-DOPA-DOPA (5%),
Fmoc-DOPA-DOPA-Lys (12.5 mg/mL), samples were prepared in either
DMSO or ethanol (12.5%) these results were compared to measurements
performed on bare glass slide.
[0179] The AFM measurment was performed by emploting force mapping
while simultaneously providing nanoscale topographical and
mechanical information about the hydrogel. Force mapping involves
generating individual force curves at discrete points on a
material, which are then used to calculate stiffness and height
values.
[0180] After overnight incubation, 10 .mu.L of each sample were
deposited on a glass slide and dried at room temperature. Force
measurements of the samples were conducted using a SiO.sub.2
colloidal probe (tip velocity 1000 nm/s, compressive force 20 nN).
To investigate the glass surface area morphology after peeling, 30
.mu.L of freshly prepared Fmoc-DOPA-DOPA-Lys solutions (12.5 mg/mL,
prepared in DMSO or ethanol as described above) were deposited
between two microscope glass slides. This construct was incubated
overnight at room temperature under 100 mg weight Finally, the
upper glass slide was peeled off and the contact area of either
slide was imaged using an Ultrasharp AFM probe (NSC21/Ti-Pt,
MikroMasch) operated in tapping mode.
[0181] Rheological Measurements
[0182] Rheological measurements for in situ-formed Fmoc-DOPA-DOPA
hydrogel were performed using an AR-G2 rheometer (TA Instruments,
New Castle, Del., USA). Time-sweep oscillatory tests in 20 mm
parallel plate geometry were conducted at 0.7% strain and 1 Hz
frequency on 200 .mu.L of fresh solution (resulting in a gap size
of about 0.6 mm), 1 min after its preparation. In order to
determine the linear viscoelastic region oscillatory strain
(0.01-100%) and frequency sweep (0.01-100 Hz) tests were conducted
45 min after diluting the stock solution with Milli-Q water. All
rheology tests were done in triplicates and averaged.
[0183] Turbidity Analysis
[0184] Turbidity analysis for Fmoc-DOPA-DOPA solutions was
conducted using freshly prepared solutions at concentrations of 2.5
and 5 mg/mL. 200 .mu.L aliquots were pipetted into a 96-well plate
and absorbance at 405 nm was measured over time, starting 45 s
after the preparation of the peptide solution. All measurements
were performed using a Biotek Synergy HT plate reader at 25.degree.
C.
[0185] Metal Deposition on Micro- and Nano-Structures
[0186] Fmoc-DOPA-DOPA
[0187] Silver reduction assay was performed with pre-prepared
Fmoc-DOPA-DOPA hydrogels at a concentration of 5 mg/mL (8.35 mM).
50 .mu.L of 13.2 mM silver nitrate were added to 500 .mu.L hydrogel
aliquots by gentle pipetting, resulting in 1.2 mM final
concentration of silver nitrate. This solution was incubated at
room temperature for several days. At different time points, 10
.mu.L aliquots were taken for TEM analysis and negative staining
was not applied. To examine silver reduction using UV-vis
spectroscopy, silver nitrate was added to a 5 mg/mL hydrogel
pre-prepared as described above and 150 .mu.L aliquots of
Fmoc-DOPA-DOPA hydrogel with or without silver nitrate were
pipetted into a 96-well UV-Star UV transparent plate (Greiner
BioOne, Frickenhausen, Germany) Spectra were collected after 5 days
using a Biotek Synergy HT plate reader over the range of 300-700 nm
and compared to blank samples of silver nitrate only, or of the
peptide only.
[0188] DOPA-DOPA
[0189] Peptide stock solution was diluted to a final concentration
of 5 mg/ml in double distilled water. Then metal salt (AgNO.sub.3)
was added to the sample and the samples were analyzed by TEM or
HR-SEM. AgNO.sub.3 concentration was determined according to the
final catechol concentration in the peptide, with a constant ratio
of [AgNO.sub.3]/[Catechol]=0.0723.
[0190] Asp-DOPA-Asn-Lys-DOPA
[0191] Silver deposition was done by preparing 15 mM peptide
solutions and removing residual peptide monomers. This was done by
centrifugation at 12,000 RPM for 15 min, discarding the supernatant
and resuspention in ultra-pure water. This procedure was repeated
once more. Samples for electron microscopy were taken as control
and the solution was centrifuged at 12,000 RPM for 15 min and
resuspended in an aqueous solution of 2.17 mM AgNO3 for 15 min
Finally, the solution was centrifuged at 9000 RPM for 10 min and
resuspended in water. Samples for electron microscopy were taken
again.
[0192] Asp-DOPA-Asn-Lys-DOPA
[0193] Silver deposition was done by preparing 15 mM peptide
solutions and removing residual peptide monomers. This was done by
centrifugation at 12,000 RPM for 15 min, discarding the supernatant
and resuspention in ultra-pure water. This procedure was repeated
once more. Samples for electron microscopy were taken as control
and the solution was centrifuged at 12,000 RPM for 15 min and
resuspended in an aqueous solution of 2.17 mM AgNO.sub.3 for 15 min
Finally, the solution was centrifuged at 9000 RPM for 10 min and
resuspended in water. Samples for electron microscopy were taken
again.
[0194] Congo Red (CR) Staining
[0195] CR staining was performed with 10 .mu.L samples of 6 mM
peptide solution. The samples were air-dried on glass microscope
slides and staining was performed by the addition of 10 .mu.L
solution of 80% ethanol saturated with Congo Red and NaCl.
Birefringence was determined using an Olympus SZX-12 Stereoscope
(Hamburg, Germany) equipped with a polarizing stage. Fluorescence
visualization was performed using Nikon Eclipse 80i epifluorescent
microscope (Kanagawa, Japan) equipped with a Y-2E/C filter set
(excitation 560/20 nm, emission 630/30 nm). Thioflavin-T (ThT)
staining was performed by adding fresh 4 mM ThT solution to an
equal volume of 6 mM peptide solution which was incubated for 3 h
prior to the addition of ThT. The resultant solution was incubated
for 3 h in the dark and 10 .mu.L samples were imaged using LSM 510
Meta confocal microscope (Carl Zeiss, Oberkochen, Germany) at 458
nm excitation and 485 nm emission.
[0196] Fourier Transform Infrared (FTIR)
[0197] FTIR spectroscopy was performed with 30 .mu.L samples of 6
mM peptide solution deposited onto disposable polyethylene IR
sample cards (Sigma-Aldrich, Israel) which were then allowed to dry
under vacuum. To achieve hydrogen to deuterium exchange, the
peptide deposits were subjected to 2 cycles of resuspension in 30
.mu.L D.sub.2O (99.8%) and drying in vacuum. Transmission infrared
spectra were collected using Nexus 470 FTIR spectrometer (Nicolet,
Offenbach, Germany) with a deuterated triglycine sulfate (DTGS)
detector. Measurements were made using the atmospheric suppression
mode, by averaging 64 scans in 2 cm.sup.-1 resolution. The amide I'
region was deconvoluted by subtracting a baseline of ultra-pure
water that was deposited on a polyethylene sample card and
subjected to two cycles of resuspension in D.sub.2O as described
above. Subtraction was performed using the OMNIC software
(Nicolet). Smoothing, second derivative calculation and
curve-fitting were then performed using the Peakfit software
version 4.12 (SYSTAT, Richmond, Calif.). For transmittance plots,
13-data-point savitzky-golay smoothing was applied to the raw
spectra using the Omnic software.
[0198] Circular Dichroism (CD)
[0199] CD spectroscopy was performed by dilution of fresh 6 mM
peptide solution in ultra-pure water to a concentration of 60
.mu.M. CD spectra were collected with a Chirascan spectrometer
(Applied Photophysics, Leatherhead, UK) fitted with a Peltier
temperature controller set to 25.degree. C., using a capped
rectangular quartz cuvette with an optical path length of 0.1 cm.
Absorbance was kept under two units during all measurements. Data
acquisition was performed in steps of 1 nm at a wavelength range
from 190-260 nm with a spectral bandwidth of 1.0 nm and an
averaging time of 3 s. The spectrum of each sample was collected
three times and a control spectrum of ultra-pure water was
collected twice. Spectra were corrected in baseline with ultra-pure
water as the blank. Data processing was done using Pro-Data Viewer
software (Applied Photophysics, Leatherhead, UK); processing and
normalization to mean residue ellipticity (MRE). To verify the
assayed solution contained characteristic assemblies, a 10 .mu.L
sample of the cuvette content was examined by TEM as described
above.
[0200] Thermal perturbation was performed using freshly made 15 mM
peptide solution diluted to a concentration of 120 .mu.M. CD
spectra were collected as described above except that the spectra
were obtained throughout temperature variation done in a stepwise
fashion up and then down. The investigated temperatures ranged over
25.degree. C.-90.degree. C. (in the following steps: 25.degree. C.,
37.degree. C., 50.degree. C., 70.degree. C., 90.degree. C.,
70.degree. C., 50.degree. C., 37.degree. C., 25.degree. C.). The
sample was allowed to equilibrate for 10 min and the temperature
was monitored by a thermocouple in the cuvette holder block. At the
end of the measurments, the cuvette content was sampled for TEM and
FTIR analysis as described above. As control, TEM and FTIR samples
were taken from an aliquot of the same solution which was not
subjected to temperature variations.
Results
Example 2
Fmoc-DOPA and Fmoc-DOPA-DOPA
[0201] Morphology Analysis
[0202] DOPA-DOPA and Fmoc-DOPA-DOPA peptides were examined under
different conditions and were found to self-assemble into ordered
nanostructures in tihe presence of ethanol and water (FIG. 2B-G).
Mactroscopically, the Fmoc-DOPA-DOPA peptide formed a
self-supporting hydrogel (FIG. 4). To gain a better insight into
the molecular organization and morphology of the formed structures,
electron microscopy was employed. Transmission electron microscopy
(TEM) analysis of both peptides revealed the formation of a tangled
fibrous network composed of flexible, elongated fibrillar
structures. The existence of twisted multistrand fibers alongside
single fibrils was observed. The DOPA-DOPA dipeptide assembled into
fibers with a cross section ranging from 20 to 50 nm (FIG. 2B, 2C)
while Fmoc-DOPA-DOPA formed narrower fibers, varying in width from
approximately 4 to 30 nm (FIG. 2D, 2E, 2F).
[0203] To characterize the morphology of the Fmoc-DOPA-DOPA
hydrogel under humid conditions, environmental scanning electron
microscopy (E-SEM) was carried out. Upon gradual dehydration of the
sample, a network of supramolecular substructures was observed
(FIG. 2G).
[0204] In another experiment, norphology characterization of the
hydrogel revealed fibrous network with fibril diameters ranging
from 20 to 100 nm (FIG. 3A-3B). The fibrils forming the network are
flexible with branching characteristics. HR-SEM samples taken
minutes or hours after the assembly process (FIG. 3C-3F) may
explain the changes in the optical properties of the sample over
time. At first, large aggregates (diameter of 20-50 .mu.m) were
observed, whereas after several hours ordered structures with
smaller diameter (20-100 nm) were seen. This observation fits a
theory suggesting that the duration of the optical transition in
Fmoc-DOPA-DOPA to the time required for the initial organization of
the molecules to undergo a physical restructuring. This
restructuring, from irregular aggregates with dimensions in the
range of the visible wavelength to ordered structures with final
diameters smaller than the visible light wavelength, causes the
changes in the optical characteristics of the solution. In contrast
to the gelation process, the decrease in turbidity was not
temperature dependent. This indicates that additional parameters,
other than temperature, affect the self-assembly process of the
hydrogel. The assumption is that spontaneous oxidation that occurs
over time has a key-role in stabilizing the formed structures.
Oxidation of DOPA to DOPA-quinone or DOPA-semiquinone can lead to
cross-linking, giving rise to solidification of the hydrogel.
[0205] Rheological Analysis
[0206] The hydrogel formed by the low molecular weight (LMW)
Fmoc-DOPA-DOPA peptide was further characterized. The viscoelastic
properties of the gel were assessed using rheological measurements.
Oscillatory strain (0.01-100%) and frequency sweep (0.01-100 Hz)
tests were conducted to determine the linear viscoelastic regime
(FIG. 4A-4B). These tests revealed that at the linear region, the
storage modulus (G') of the hydrogel is more than one order of
magnitude larger than the loss modulus (G''), a rheological
behavior that is characteristic of elastic hydrogels. As shown in
FIG. 4C, the plateau storage modulus of the hydrogel was found to
be modulated with a high dynamic range of .about.20 Pa to .about.5
kPa, corresponding to the final concentration of the peptide.
Furthermore, the gelation kinetics was found to be temperature
dependent (FIG. 4D), as gelation was highly decelerated at
4.degree. C. compared to 25.degree. C. or 37.degree. C. At higher
temperatures (25.degree. C. or 37.degree. C.), the storage moduli
of the hydrogels were approximately 40 fold higher than the storage
modulus of hydrogels formed at 4.degree. C. The gelation process of
Fmoc-DOPA-DOPA was also accompanied by a change in the optical
properties of the sample, transforming from a turbid viscous
solution to a semi-transparent hydrogel (FIG. 4E-left panel). When
the 2.5 mg/mL sample was observed macroscopically, the solution
cleared within minutes, corresponding to the formation of the
hydrogel (FIG. 4E-center panel). In contrast, in the case of the 5
mg/mL, although the solution cleared within minutes gelation
occurred after longer periods of time (FIG. 4E-right panel).
High-resolution SEM (HR-SEM) analysis of the 5 mg/mL samples taken
minutes or hours after the assembly process (FIG. 4F) indicated
that large aggregates are present at the initial stage when the
solution is turbid, whereas after several hours, ordered structures
with much smaller diameter appear, corresponding to a considerably
clearer solution. This observation is also in line with a previous
hypothesis put forward by the present inventors linking the optical
transition from turbid to transparent in Fmoc-protected hydrogel
preparation to the ultrastructural organization over time [28].
This restructuring, from irregular aggregates with dimensions
similar to or higher than the wavelengths in the visible spectrum
to ordered structures with final diameters much lower than these
wavelengths, results in the change of the scattering properties of
the solution.
[0207] Adhesion Measurements
[0208] The adhesion of Fmoc-DOPA-DOPA adhesion to silicon oxide was
intially estimated as described in Example 1 (Adhesion
measurements-Embodiment 1) where a peptide solution at two
different concentrations (2 mg/ml and 5 mg/ml) were deposited onto
a glass slide and imaged with the AFM (FIGS. 5A and 5B,
respectively). The adhesion force was found for 2 mg/ml (5
locations) and 5 mg/ml (3 locations) by measuring the interaction
of the AFM tip and the surface. The adhesion of the AFM tip to the
Fmoc-DOPA-DOPA sample ranged between 28 to 87 nN as summarized in
the tables (FIG. 5C-5D).
[0209] The adhesion of the peptide to silicon oxide was measured as
described in Example 1 (Adhesion measurements-Embodiment 2). AFM
measurements were initially performed on a bare glass slides and a
(20 .mu.m.times.20 .mu.m) force map (FIG. 6A) was generated. The
adhesion force map on the glass slide (FIG. 6B) resulted in an
adhesion histogram in which the mean force is 7.+-.1.2 nN.
[0210] Fmoc-DOPA-DOPA peptide (5 mg/ml) was deposited onto a glass
slide and imaged with the AFM in tapping mode (with a ultra sharp
MicroMasch AFM probe), with a scan size of 3.times.3 .mu.m.sup.2
(FIG. 7A). This revealed the presence of bulk fibrous structures.
The adhesion force of the AFM tip (SiO.sub.2 colloidal probe) to
the Fmoc-DOPA-DOPA peptide was performed by generating a
20.times.20 .mu.m.sup.2 force map (FIG. 7B) to the AFM. From the
adhesion histogram (FIG. 7C) the mean force was calculated to be
36.+-.11 nN. The tip velocity was 1000 nm/s and the compressive
force was 20 nN.
[0211] Redox Properties
[0212] The catechol group of the DOPA moiety has redox properties
that enable the spontaneous reduction of metal cations into neutral
metal atoms resulting in metal nanoparticles. The Fmoc-DOPA-DOPA
based hydrogel was tested for having the inherent property to
spontaneously form silver particles from silver nitrate. During the
assembly process of Fmoc-DOPA-DOPA, the catechol functional group
could either be directed towards the inner core of the assembled
structure or towards the solution. It was hypothesized that DOPA
groups that are not facing towards the hydrophobic core of the
structures will be able to react with the silver salt to form
silver particles that can be easily detected using electron
microscopy. Moreover, the formation of Ag is accompanied by a color
change resulting in an absorbance peak centered at .about.400 nm
that can be easily be observed by the naked eye.
[0213] The addition of silver nitrate solution to a pre-prepared
Fmoc-DOPA-DOPA hydrogel led to a change in the hydrogel color from
semi-transparent to dark brown over a period of several hours to a
few days (FIG. 8A). UV-vis spectra taken 5 days after the silver
nitrate was added to the hydrogel revealed an increase in the
absorbance above 300 nm. The broadband increase in the absorption
in this area could be due to scattering by silver nanoparticles and
DOPA oxidation.
[0214] The ionic silver reduction process was further monitored by
TEM. A slow, gradual transition from the formation of local silver
nanoparticles nuclei to the formation of a continuous silver layer
was observed (FIG. 8B). Distinct formation of silver nanoparticles
was observed after short incubation or at a low concentration of
the peptide. However, after a longer incubation at a high peptide
concentration, the seamless coating of the peptide assemblies was
clearly observed (FIG. 8C). Such uniform and continuous coating is
unique and is most likely the result of both the slow reduction
kinetics, as discussed above, and the high density of catechol
groups presented by the assemblies.
[0215] The reaction of DOPA-DOPA dipeptide (FIG. 9A) with silver
nitrate had resulted in the formation of silver particles (FIG.
9B-9C). This reduction of the silver-nitrate (AgNO.sub.3) can also
be seen in the appearance of an absorbance peak (FIG. 10A) at 390
nm (reaction at pH=7) and at 400 nm (reaction at pH=8). A zoom-in
of the absorbance peaks (FIG. 10B) further reveals the differences
in absorption values in the presence and absence of silver
nitrate.
Example 3
Fmoc-DOPA
[0216] Morphology Analysis
[0217] AFM and light microscpy were both employed to study the
self-assembly properties of Fmoc-DOPA peptide. It was found that
under different conditions ordered spheres-like structures were
formed with diameters ranging between dozens of nm to microns.
Measurements were conducted on samples of Fmoc-DOPA at different
concentrations (2 or 5 mg/ml), pH (5.5 or 8.7) and in the absence
or presence of Fe.sup.+3. The hypothesis is that both basic pH and
Fe.sup.+3 ions may contribute to the adhesion of Fmoc-DOPA.
[0218] In general all experiments were performed with water (pH
5.5). In some cases, the water medium was pre-adjusted to the
specific pH mentioned by the addition of diluted NaoH solution. Due
to the non-buffered conditions, after addition of the acidic
peptides, the over all pH of the system was acidic. Examination of
5 mg/ml Fmoc-DOPA at acidic pH (.about.5.5) showed sparse spheres
at varied sizes (FIG. 11A-11C). Measurements of 5 mg/ml Fmoc-DOPA
revealed a dense layer of spheres with variying sizes, having
diameters ranging between dozens of nano-meters to microns (FIG.
12A-12E). A sample of 5 mg/ml Fmoc-DOPA at with the presence of
Fe.sup.+3 also displayed a dense layer of spheres at varied sizes.
Fmoc-DOPA at lower concentration (2 mg/ml) also exhibited diversity
in structures sizes, with a population composed of large truncated
spheres and small spheres (FIG. 13A-13C).
[0219] Adhesion Measurements
[0220] Several force-distance curves have been taken, as described
in Example 1 (Adhesion measurements-Embodiment 1), to measure the
adhesion of the Fmoc-DOPA peptide under the various conditions
described above. The results of the adhesion measurements for the 5
mg/ml Fmoc-DOPA at acidic pH (5.5) sample are summarized in the
table in FIG. 11D. The adhesion of the AFM tip to the Fmoc-DOPA
peptide was found to range between 23 nN to 93 nN. Adhesion
measurements for the 5 mg/ml Fmoc-DOPA sample ranged between 24 to
97 nN (table in FIG. 12F). Adhesion measurements for the sample
with the presence of Fe.sup.+3 ranged between 40 nN to 210 nN
(Table in FIG. 12G). Adhesion measurements for 2 mg/ml Fmoc-DOPA
sample resulted in values ranging between 13 nN to 50 nN (table in
FIG. 13D).
[0221] A 10.times.10 .mu.m.sup.2 topography (FIG. 14A) and a
20.times.20 .mu.m.sup.2 force map (FIG. 14B) images of the
deposited Fmoc-DOPA (1 mg/ml) were taken (Adhesion
measurements-Embodiment 2) followed by the construction of an
adhesion-force histogram (FIG. 14C). It was found that the mean
adhesion force was 31.+-.10.6 nN, a similar value to that found for
the Fmoc-DOPA-DOPA peptide using the same measurement. The tip
velocity was 1000 nm/s and the compressive force was 20 nN.
Example 4
Fmoc-DOPA-DOPA-Lys
[0222] It was hypothesized that the incorporation of a lysine
residue into the DOPA-containing peptide assemblies would
contribute to cohesion and thus indirectly improve its adhesion.
Moreover, lysine residues may also contribute to adhesion via ionic
bonding to negatively charged surfaces. Therefore, the
Fmoc-DOPA-DOPA-Lys (FIG. 15A-left panel) protected tripeptide was
designed and tested.
[0223] Morphology Analysis
[0224] Upon examination of the protected tripeptide under the
conditions applied to the two peptides studied initially (DOPA-DOPA
and Fmoc-DOPA-DOPA), the Fmoc-DOPA-DOPA-Lys peptide was also found
to self-assemble into well-ordered fibrillar structures. However,
in contrast to the fibers formed by the former, the fibers
assembled by the Fmoc-DOPA-DOPA-Lys were narrower, with an
approximated width of less than 10 nm. Moreover, the fine fibers
were only formed by dissolving the peptide to higher concentrations
of (1.25 wt % versus 0.5 wt %) as can be seen in the center panel
of FIG. 15A. Fmoc-DOPA-DOPA-Lys was also found to self-assemble
into ordered nanostructures in the presence of dimethyl sulfoxide
(DMSO) and water (FIG. 15A-right panel), forming assemblies that
also displayed a high degree of ultrastructural similarity to the
Fmoc-DOPA-DOPA structures.
[0225] Adhesion Measurements
[0226] To quantify the adhesive forces of the tripeptide sample to
the glass surfaces, AFM measurements were taken, as described in
Example 1 (Adhesion measurements-Embodiment 2). Specifically, the
adhesion of the structures to a silicon oxide (SiO.sub.2) colloidal
probe was assessed by employing force-distance measurements. In
comparison to the very low adhesion of the AFM probe to bare glass,
a glass surface covered with Fmoc-DOPA-DOPA-Lys tripeptide
assemblies displayed significant adhesive forces. Whereas the
adhesion of the tip to bare glass was less than 10 nN (FIG. 6B), it
was found that the adhesion of the Fmoc-DOPA-DOPA-Lys to the glass
was calculated to be more than 214 nN when prepared in ethanol
(FIG. 15B) and 300 nN (FIG. 15C) when prepared in DMSO.
[0227] Macroscopically, it was observed that this protected
tripeptide forms viscoelastic glue capable of adhering two glass
slides. Under both preparation conditions (i.e. ethanol and DMSO),
the sample displayed macroscopic adhesive properties, capable of
gluing together two glass slides. It should be noted that this
gluing phenomenon, in the presence of DMSO, showed recovery
behavior: after peeling the glass of from each other they were able
to be re-joined. Interestingly, this was not the case when ethanol
was used for the preparation of Fmoc-DOPA-DOPA-Lys solutions. When
this procedure was repeated with an ethanol-prepared tripeptide
sample, the sample lost its adhesive properties after peel forces
were applied.
[0228] To understand the basis for the recovery differences between
the DMSO and ethanol tripeptide samples, after the two glass slides
were glued together we peeled off the glass slide and examined the
exposed area. AFM analysis of Fmoc-DOPA-DOPA-Lys in ethanol after
peeling (FIG. 15D) exhibited unidirectional fine fibrous
structures. In contrast, AFM analysis of Fmoc-DOPA-DOPA-Lys in DMSO
after peeling (FIG. 15E) revealed the presence of twisted spheres
that were unidirectionally retracted.
Example 5
DOPA-Phe-Phe
[0229] The self-assembly ability of DOPA-Phe-Phe was examined
Examination of the DOPA-Phe-Phe peptide revealed that this peptide
assembles into sphere-like particles, with diameters ranging
between 30 to 100 nm, when it is dissolved in HFIP (100 mg/ml) and
then diluted in water to a final concentration of 2 or 5 mg/ml
(FIG. 16A-16F). In addition, DOPA-Phe-Phe (50-100 mg/ml), in the
presence of 100% HFIP, was found to form highly dense horizontal
aligned ribbon-like structures with diameters ranging between 30 to
100 nm. These structures were found to be filamentous, branched and
flexible (FIGS. 17A-17G and FIGS. 18A-18C). 100 mg/ml DOPA-Phe-Phe
formed dense tube-like ordered structures Examination of individual
tube-like structures formed by 50 mg/ml DOPA-Phe-Phe revealed, at
the 3D imaging, wall-like structures with cratered surface (FIGS.
19A-19E). The spatial arrangement of the peptides is probably due
to the rapid evaporation of HFIP.
[0230] Adhesion Measurements
[0231] 100 mg/ml DOPA-Phe-Phe sample adhesion was found to be in
the range from 40 nN to 121 nN (FIG. 18D). Adhesion measurements of
50 mg/ml DOPA-Phe-Phe sample were between 29 to 74 nN (FIG.
19F).
[0232] In summary, the aforementioned examples show that the
substitution of phenylalanine with DOPA in the known
self-assembling peptide motif FF yields novel self-assembling
peptides that are able to form ordered supramolecular structures
substantially decorated with catechol functional groups. Due to the
intrinsic properties of the catechol group, the obtained
supramolecular structures can be used as new multifunctional
platforms for various technological applications. Upon the
incorporation of additional lysine residue containing 6-amine,
significant adhesion was obtained, possibly due to electrostatic
interactions between the protonated amine and negatively charged
oxide surface. The remarkable seamless silver deposition reflects
the tendency of the dense catechol array to facilitate coating
rather than adhesion. The properties of this deposition are unique,
as compared to any known electroless metal coating of biological or
polymer nano-assemblies and should prove very useful in the
templating of inorganic materials on organic surfaces at the
nano-scale for various applications, e.g., formation of
anti-bacterial hydrogels, among others.
Example 6
Pentapeptides DOPA-Containing Fibrillar Assemblies
[0233] Amyloid fibrils self-assemble through molecular recognition
facilitated by short amino acid sequences found in amyloidogenic
proteins or polypeptides, which were identified as minimal
amyloidogenic recognition modules. These modules can serve as
initiators and facilitators of aggregation and vary between
amyloidogenic proteins and polypeptides. By employing a
reductionist approach, in vitro studies utilizing short synthetic
peptides as model systems led to the discovery of minimal
recognition modules in numerous amyloidogenic proteins and
polypeptides. One such module was identified in human calcitonin
(hCT), a 32-residue polypeptide hormone which plays a role in
calcium-phosphate homeostasis. hCT can form amyloid fibrils in vivo
and the fibrils were implicated in the pathogenesis of medullary
thyroid carcinoma. The sequence Asp-Phe-Asn-Lys-Phe (SEQ ID. No.
2). was previously identified as the minimal amyloidogenic
recognition module of hCT [15]. This pentapeptide, spanning
residues 15-19 of hCT, forms amyloid fibrils in vitro at neutral pH
in aqueous solutions with remarkable similarity to the fibrils
formed by the full-length hCT.
[0234] Self-assembly of amyloid-like structures has been a subject
of much interest in nanobiotechnology. Due to their ability to
self-assemble into ordered nanostructures that may also be
chemically and biologically functionalized, amyloidogenic peptides
are regarded as promising building blocks for various
nanobiotechnological applications.
[0235] As demonstrated herein, a short synthetic peptide, the
minimal amyloidogenic recognition module of hCT (SEQ ID ID No. 1)
(FIG. 20A), was designed to contain two DOPA moieties, substituting
the phenylalanine residues, resulting in a unique building block,
the Asp-DOPA-Asn-Lys-DOPA pentapeptide (FIG. 20B). This
pentapeptide retains the ability to spontaneously self-assemble in
vitro into amyloid-like fibrillar assemblies in simple aqueous
solutions. The obtained assemblies displayed structural properties
characteristic of amyloids as well as characteristics of
DOPA-containing polypeptides. Functional assessment of the
assemblies suggested redox activity and demonstrated the
applicative potential of this novel nanobiomaterial.
Morphology Characterization
[0236] Lyophilized Asp-DOPA-Asn-Lys-DOPA peptide was dissolved in
aqueous solutions followed by the application of bath-sonication
for 10 min Concentrations of 100 .mu.M to 15 mM were tested and a
viscous turbid solution was obtained in all cases. Samples were
examined by TEM (FIGS. 21A-21B) and E-SEM (FIG. 21C). A network of
fibrillar assemblies was detected and the observed fibrillar
assemblies were mostly linear, unbranched and extending to the
length of micrometers. The width of individual fibrillar assemblies
varied from 15 to 85 nm and lateral bundling of the assemblies was
observed. Such morphological features are common to amyloids and
amyloid-like structures.
Secondary Structure Analysis
[0237] A hallmark of the amyloid cross-.beta. structure is
apple-green birefringence of the dye Congo Red (CR) under polarized
light when bound to amyloid fibrils. This can be supported by CR
fluorescence, which gives red-orange emission (616 nm) upon green
excitation (510-560 nm). When Asp-DOPA-Asn-Lys-DOPA samples were
dried and stained with CR, apple-green birefringence (FIG. 22A) and
red-orange fluorescence (FIG. 22B) were observed while virtually
none were observed in control samples of CR only or of the peptide
without staining. FIG. 22C represents brightfield image
corresponding to the fluorescence microscopy micrograph (scale bars
represent 100 .mu.m).
[0238] Further analysis was performed using transmission Fourier
transform infrared (FTIR) spectroscopy. Samples of 6 mM
Asp-DOPA-Asn-Lys-DOPA solutions were dried, subjected to
hydrogen-to-deuterium exchange and analyzed. The second derivative
of the amide I' region (1600-1700 cm-1) was curve-fitted and
component bands were assigned to secondary structure elements (FIG.
23A). Distinct peaks were found at approximately 1627 and 1679
cm-1, which are attributable to the presence of .beta.-sheet
structures. The combined presence of a low component around 1630
cm-1 and a weaker, high component around 1680 cm-1 is consistent
with an antiparallel .beta.-sheet structure. Indeed, an
antiparallel .beta.-sheet structure, albeit with slightly different
peak positions, was previously reported for fibrils formed by the
unmodified hCT minimal recognition module. A third peak, at 1659
cm-1, can be attributed to .alpha.-helical structures. It was
reported that such structures appear in solutions of the unmodified
hCT minimal recognition module in a concentration-dependent
manner.
[0239] To examine the secondary structure of Asp-DOPA-Asn-Lys-DOPA
in solution, freshly made 6 mM solutions were diluted to a final
concentration of 0.15 mM and circular dichroism (CD) spectroscopy
in the far-UV region was performed at 25.degree. C. Weak negative
ellipticity was observed at approximately 235-250 nm, followed by a
weak positive shoulder around 230 nm, a positive maximum at 209 nm
and a negative maximum at 194 nm (FIG. 23B). A remarkably similar
CD spectrum has been previously reported for the highly-polymerized
poly(L-Lys-L-Lys-L-Lys-L-DOPA) sequential polypeptide in water at
25.degree. C. A negative maximum near 195 nm indicated that the
sequential polypeptide adopted a random coil conformation under
these conditions, whereas the positive ellipticity around 220 nm
was attributed to transitions of the catechol side-chain and not to
secondary structure elements. The CD spectrum of
Asp-DOPA-Asn-Lys-DOPA therefore suggests that the peptide adopts a
random coil conformation in solution. This result is surprising on
several counts. First, a characteristic random coil band in the
Amide I' region was not detected by FTIR spectroscopy. Second, TEM
examination of solution samples taken from the CD cuvette at the
end of the measurement contained ample amyloid-like fibrillar
assemblies as described above (data not shown); since no additional
CD bands were detected and since the ultrastructure was not lost,
the CD measurement seems to represent the secondary structure of
the peptide while being part of the ultrastructure. Third, the CD
spectrum of the unmodified hCT minimal recognition module under the
same conditions and at similar concentrations showed an
.alpha.-helical structure.
[0240] To further elucidate the structural properties of
Asp-DOPA-Asn-Lys-DOPA, temperature-dependent CD was performed.
Freshly made 6 mM peptide solutions in water were diluted to a
final concentration of 0.15 mM and CD spectra were collected during
a stepwise increase in temperature from 18.degree. C. to 90.degree.
C. and a subsequent stepwise decrease to 18.degree. C. Throughout
this process, the spectral profile retained its distinct features
(FIG. 24A). However, intensity loss was observed as the temperature
increased, with a significant loss occurring near the 209 nm band
and in the 235-250 nm region. This effect seemed irreversible as
only little intensity gain was observed upon temperature decrease.
Subsequent FTIR analysis of the CD cuvette content showed no bands
in the amide I' region while a control sample, taken from the same
solution and kept at room temperature, displayed the characteristic
Asp-DOPA-Asn-Lys-DOPA FTIR spectrum with peak positions at 1627 and
1659 cm-1 (FIG. 24B). Moreover, in a TEM examination, the solution
that was subjected to temperature variations did not contain
assemblies (FIG. 24B1--insert) while the control solution contained
fibrillar assemblies (FIG. 24B2--insert). Taken together, these
results suggest that the ultrastructure is impaired by elevated
temperatures in a process accompanied by aggregation and
sedimentation of the peptide.
[0241] Since the first significant change in the CD signal was
observed when the temperature was increased from 25.degree. C. to
37.degree. C., we sought to examine the ultrastructural effect of
subjecting the assemblies to this particular temperature. To this
end, a peptide solution at a concentration of 6 mM was allowed to
self-assemble at room temperature for four days then incubated
overnight at 37.degree. C. TEM samples were taken from this
solution immediately after incubation as well as after 8 h of
recovery at room temperature. A third sample was taken from a
solution aliquot incubated at room temperature as control. While
the aliquot incubated at room temperature contained a dense network
of fibrillar assemblies with characteristic morphology (FIG. 25A),
the aliquot incubated at 37.degree. C. contained fewer assemblies,
from which fine fibrillar protrusions were extending (FIG. 25B).
This morphological transition correlates with the CD signal
intensity loss upon heating to 37.degree. C. Moreover, in
accordance with the CD results, this transition does not seem to be
reversible as the characteristic morphology was not fully retained
following recovery in room temperature (FIG. 25C). Since the
morphological transition was accompanied by a decrease in the
abundance of assemblies, it follows that this ultrastructural
reorganization process leads to the aggregation and sedimentation
of the assemblies.
Functional Examination
[0242] The ability of Asp-DOPA-Asn-Lys-DOPA assemblies to reduce
ionic silver was examined. An aqueous peptide solution was produced
by means of repeated pelleting in water to remove any peptide
monomers. Subsequently, AgNO3 solution was used for resuspention of
the pellet and the solution was incubated for 15 min then
re-pelleted and resuspended in water. TEM examination of the
resultant solution revealed significant deposition of silver on the
fibrillar assemblies which appeared as dark nanometric clusters
(FIG. 26A), while this was not observed in a control solution to
which AgNO3 was not added. Furthermore, the clusters seemed to
selectively deposit on the assemblies compared to the background.
Similar results were obtained in an E-SEM examination, with the
clusters appearing in white (FIG. 26B). The results show that
Asp-DOPA-Asn-Lys-DOPA assemblies possess the ability to reduce
ionic silver while retaining their ultrastructure in solution.
[0243] In conclusion, as demonstrated herein, a DOPA-incorporated
pentapeptide inspired by a minimal amyloid recognition module can
self-assemble into an amyloid-like supramolecular polymer of
fibrillar nature in simple aqueous solutions. The assemblies formed
were investigated by electron microscopy, amyloidophilic dyes and
spectroscopic methods. The investigation revealed that the
supramolecular polymer formed is endowed with characteristics of
both amyloids and DOPA-containing polypeptides. Furthermore, the
ability to reduce ionic silver while maintaining the
ultrastructural integrity has demonstrated the applicative
potential of this novel nanobiomaterial.
[0244] While the present invention has been particularly described,
persons skilled in the art will appreciate that many variations and
modifications can be made. Therefore, the invention is not to be
construed as restricted to the particularly described embodiments,
and the scope and concept of the invention will be more readily
understood by reference to the claims, which follow.
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Sequence CWU 1
1
2132PRTHomo sapiens 1Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr
Tyr Thr Gln Asp Phe 1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr
Ala Ile Gly Val Gly Ala Pro 20 25 30 25PRTArtificial
SequenceSynthetic peptide 2Asp Phe Asn Lys Phe 1 5
* * * * *